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Let's make Double Haploids

Only Ornamental

Spiritually inspired agnostic mad scientist
Veteran
Err....Earthy, this is the doubled haploid thread, not the mutation breeding one ;) .
I wouldn't try arsenic, bismuth, or any other toxic heavy metal for that purpose anyway. I wouldn't even want to consume an extract of plants treated in such a way cause extraction ain't a good enough protection ;) .

Oryzalin might indeed come in handy when making DH.
 

Stan G.

Member
I was totally absorbed in this thread! What a great topic. I find it very interesting Sam hasn't threw his two cents in, seems right up his alley. Are chemicals like Colchicine the only way to induce dub hap?:tiphat:
 

rykus

Member
I remember a lot of talk of using colchicine for getting mutations in the early 90's when I first was growing... I also remember some speculation a few varieties that did not breed true where suspected of being worked with such and as a result not breeding well because the other sets of genes have nothing to attatch too...

I don't understand it well enough myself but are you not doubling the potential genes to be able to isolate and stabalise desirable traits?

Is this the same tech that brought strawberries from 2 to 8 possible gene combos or something like that? Vague memories...

Cool discussion! I feel stoned for the first time all week,lol
 

Only Ornamental

Spiritually inspired agnostic mad scientist
Veteran
...Are chemicals like Colchicine the only way to induce dub hap?:tiphat:
Not exactly... For induction of haploid plants one does things like growing anthers in a Petri dish and treat them with heat, cold, or other stresses and not only chemicals (if ever). There are huge differences regarding the needed stimuli not only between species but also between varieties and cultivars. In the next step, the doubling of the chromosomes, colchicine and other spindle toxins such as oryzalin are often a must. It's not about doubling single genes or inducing mutations, but simply the chromosome doubling naturally happening after pollen and egg (both are haploid BTW) fuse.
 

Iachdo

New member
Is this the same as protoplast fusion or different? I have just started learning about these thing and am very interested in continuing my studies but pardon me if I am being ingnorant.
 

Only Ornamental

Spiritually inspired agnostic mad scientist
Veteran
No, it's not ;) . Doubled haploids means dividing the diploid chromosome set in half and then copying it to get back to a diploid state wherein the cell is now 100% "inbred". Protoplasts are plant cells without cell wall (i.e. similar to animal cells which only have a cell membrane) and fusing two such cells together by using some sort of stress. This results in a new cell now containing twice as many chromosomes (e.g. tetraploid). This works fairly well with plant cells and is used to "cross" two completely different species.
 

Iachdo

New member
Word. You could cross species but then in the same regard you could just cross two different strains without having to herm a plant. What I’m not sure about is would that new plant still be just as unstable as the herm plant. There are studies supporting crop improvements in tomato’s and potato’s by using this method. The main reason cannabis is so fascinating is the wide variety of different horticultural techniques that only work on a certain species of plant but can also be applied to cannabis. I’m planning on having some fun this year...
 
Y

Yard dog

Not exactly... For induction of haploid plants one does things like growing anthers in a Petri dish and treat them with heat, cold, or other stresses and not only chemicals (if ever). There are huge differences regarding the needed stimuli not only between species but also between varieties and cultivars. In the next step, the doubling of the chromosomes, colchicine and other spindle toxins such as oryzalin are often a must. It's not about doubling single genes or inducing mutations, but simply the chromosome doubling naturally happening after pollen and egg (both are haploid BTW) fuse.

Whilst colchicine will work you can use the likes of nitrous oxide, acenaphthene, 8-Hydroxyquinoline, p-fluorophenylalanine, Chloramphenicol (ie eye drops) to name a few sources...
Even if one is to look at just mutation breeding though I'm not sure there's any benefit to cannabis doing so, you could vastly speed up the process with DH's as you can fix the mutation in the 1st gen after the treatment.. (the mutant bit is more aimed at Rox/Mate dave ;))
 
R

Rox

Whilst colchicine will work you can use the likes of nitrous oxide, acenaphthene, 8-Hydroxyquinoline, p-fluorophenylalanine, Chloramphenicol (ie eye drops) to name a few sources...
Even if one is to look at just mutation breeding though I'm not sure there's any benefit to cannabis doing so, you could vastly speed up the process with DH's as you can fix the mutation in the 1st gen after the treatment.. (the mutant bit is more aimed at Rox/Mate dave ;))


What? If you have something to say then quote me and say it, don't sit at your keyboard and launch attacks by proxy
 

cannagro

New member
High Tony

I've been trying to get my head around this & my head is spinning:laughing: managed to get as far as understanding that a double haploid is a diploid as it carries 2 sets of chromosomes..Yeah? Or is a double haploid something else?

So if i am right, a diploid is identical to its parent? A haploid is a combination of both but not unique to either parent..

So if this diploid under goes Apomixis then..BINGO! Instant homozygous seeds...

I'm so excited at this prospect, assuming I have understood this correctly?

Anybody care to share some pictures?

Thanks

So a double haploid IS a diploid BUT a diploid is not always a double haploid. Let me try to explain.... A pollen grain from the male plant is a haploid that carries 1/2 the chromosomes that will make up the final plant. The egg cell in the female has the other half of the chromosomes. We will focus on the male plant because generally double haploids are made from pollen grains.

Basic mendelian genetics says each gene has 2 possible alleles, a dominant and a recessive allele which segregate when the haploid cells are created. This means that each pollen grain contains either a dominant allele or a recessive allele. This is bad for breeders because it adds variability into their breeding program. Breeders want to know the exact alleles that are being crossed. Example: A plant with the genotype AaBbCcDd segregates into the following possible combinations of alleles in the pollen grains: AbCD, abcd, ABCD, aBCD, AbcD...... If you take one of these pollen grains (ABCD)and use it to make a double haploid then the genotype of the double haploid plant (diploid) is AABBCCDD. You take the chromosomes in the pollen grain and double them to create the proper number instead of fertilizing an egg cell. This is very good for breeders because then everytime this plant produces a seed it gives off a dominant allele and dominant alleles are generally good for yield and other traits.

So in the end... A double haploid has 2 identical sets of chromosomes and a normal diploid has 2 unique sets of chromosomes.

Hope this helps.
 

Know to grow

New member
Haploids done?

Haploids done?

Hi, I am new to this forum and re-entering the world of Cannabis. I did do some selection work in the 80's. Now looking at breeding work. I am curious if any of you has any information on success in working with haploids in Cannabis.
I have made them in several crops and I am looking for some information in Cannabis. Maybe there is more success with ovu-culture?
Any people breeding who are interested in some chatting on this, I would be very interested to exchange ideas.
 

englishrick

Plumber/Builder
Mentor
ICMag Donor
Veteran
Ohhh,, geeezz

I'm about to do meristem culture,, later protoplast and possibly somatic fusion,, later i wool definitely want to do this work,,,

Incredible sruff Tony
 

brluban

Member
Alright, So... Double haploids are really neat and are deffinetely in the future of breeding. However, creating DH (double haploid) lines is a lot more complex than just using some mutagen causing hormone spray. DH lines in corn require a very specific gene which allows for the male to "pollinate" the female without actually undergoing sexual reproduction. This results in a (small proportion of) seed (have to be tagged) only possessing the genotype from the "female mother" i.e. haploid. These seeds are then germinated and treated with a chemical which double the zygosity of the individual, making a "double haploid" with 100% homozygosity, ie two copies of whichever genes were inherited from mom. This process allows for a breeder to get perfectly homozygous lines after just 1 generation as opposed to the traditional 7. Triggered by this extreme reduction in breeding time, nearly all major private companies have started utilizing DH lines. BUUTT, after nearly a decade and a half of intensive research from all major private and public breeding institutions, haploid induction rate (HIR) for seed have just now reached about 7% of total seed produced, and the gene of interest has been identified to somewhere on Chromosome 5 (not very precise) (It's gonna be different for bud) . So, if one wanted to do undrgo DH breeding with cannabis, they would have to 1) sift through hundreds and hundreds of wild type lines, using them all for pollination, looking for 1 that has the gene (if the gene even exists in cannabis) that creates a ~2-3% of haploid runt seeds. They would then have to 2) identify the trait of interest. and , work the DH line to be able to mark the seeds (not sure how this works) and hopefully make upgrade to about 7-10% HIR, so you're getting more than a couple haploid seeds per run. At that point, they could use the male DH to cross with a female and cut down 5-6 generations.

I'm sure at some point, DH will be the future of cannabis breeding, as today it is the reality of maize breeding. But before it becomes mainstream we're gonna have to invest an obscene amount of money and molecular breeding effort. TBH, if this is an option you're serious in, than the 7 traditional generations to get to a ~100% homozygous line should seem like nothing
 
Last edited:

zif

Well-known member
Veteran
Although it'd be great if someone found an inducer male, at the moment most folks are focused on anther culture. That avoids most of the issues you've highlighted from corn.

In particular, any plants produce are *definitely* either haploid or doubled haploids, depending upon whether cannabis tends to spontaneously double. There's no sifting on that front - only with respect to genotypes of interest.

Alright, So... Double haploids are really neat and are deffinetely in the future of breeding. However, creating DH (double haploid) lines is a lot more complex than just using some mutagen causing hormone spray. DH lines in corn require a very specific gene which allows for the male to "pollinate" the female without actually undergoing sexual reproduction. This results in a (small proportion of) seed (have to be tagged) only possessing the genotype from the "female mother" i.e. haploid. These seeds are then germinated and treated with a chemical which double the zygosity of the individual, making a "double haploid" with 100% homozygosity, ie two copies of whichever genes were inherited from mom. This process allows for a breeder to get perfectly homozygous lines after just 1 generation as opposed to the traditional 7. Triggered by this extreme reduction in breeding time, nearly all major private companies have started utilizing DH lines. BUUTT, after nearly a decade and a half of intensive research from all major private and public breeding institutions, haploid induction rate (HIR) for seed have just now reached about 7% of total seed produced, and the gene of interest has been identified to somewhere on Chromosome 5 (not very precise) (It's gonna be different for bud) . So, if one wanted to do undrgo DH breeding with cannabis, they would have to 1) sift through hundreds and hundreds of wild type lines, using them all for pollination, looking for 1 that has the gene (if the gene even exists in cannabis) that creates a ~2-3% of haploid runt seeds. They would then have to 2) identify the trait of interest. and , work the DH line to be able to mark the seeds (not sure how this works) and hopefully make upgrade to about 7-10% HIR, so you're getting more than a couple haploid seeds per run. At that point, they could use the male DH to cross with a female and cut down 5-6 generations.

I'm sure at some point, DH will be the future of cannabis breeding, as today it is the reality of maize breeding. But before it becomes mainstream we're gonna have to invest an obscene amount of money and molecular breeding effort. TBH, if this is an option you're serious in, than the 7 traditional generations to get to a ~100% homozygous line should seem like nothing
 

englishrick

Plumber/Builder
Mentor
ICMag Donor
Veteran
I'd go anther culture,, that was my proposal,, but it does make you think about samples prior to breeding
 
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