Doubled haploids

[HaploidDoubler]

Hi all, I am an undergraduate assistant researcher in a plant genetics lab. (Full disclaimer: this does not mean I am a geneticist!) Our lab's chief research is phylogeny of brassica genus. My job just so happens to be development of doubled haploid lines of brassica, focusing on brassica in the trianglle of U (i.e nigra, carinata, oleracea, napus, rappa, juncea) these guys are important because they are at model for studying polyploidy in plants. Anyway it's done by selecting buds that are likely to contain microspores (think of them as gamete stem cells!), then they are treated with mercury chloride to clean the buds without damaging the outer layer of the bud. This is done in very aseptic conditions, over a 5 hour protocol, one mold spore or bacterium will ruin everything! after 10 minutes, the HgCl2 is flushed out, I mash up the buds, put them through a refrigerated centrfuge several times to get the microspores to pellet. then I plate them in a liquid media. Then they are "heat-shocked" at 30 C for 24 hrs to arrest meiosis (inhibiting spindle formation I think, don't quote me!) and facilitate haploid doubling, than incubated at 21 C for ~21 days. If I'm lucky, embryos will form, i grow those on a shaker in the dark for a week maybe. I grow them on solid agar media with a cocktail of hormones and MS media until they form a callus (a lumpy plant-tumor thing), and put it on MS with GA3 hormone and they will hopefully grow shoots, that I grow essentially as if it were a clone. What I'm doing is neat, I'm doing it on other species of brassica as well, stuff there aren't even methods papers on yet! Its essentially like "super-selfing," not sure what caveats would exist in a monoecious plant like Cannabis for something like this, but I would like to figure that out! It would dramatically reduce time needed to make true-breeding lines I would think, but I don't want to make baseless claims. Also, you do sometimes get cool mutants! for example, a mutation that caused the leaves on a botrytis (cauliflower) plant to wrap around the petiole like an elm leaf, and possibly a Siamese twin broccoli. here's a paper on this too! https://www.researchgate.net/public...rd_cauliflower_Brassica_oleracea_var_botrytis

 

[Coughie]

Subbing,
I need to read this thread.

 

[zif]

In contrast, if someone finds (and shares) an inducer male, you could pollinate a female and then simply sprout haploid seeds! In many species these actually show spontaneous chromosome doubling, so you have doubled haploids (I like to think of them as S∞) in one step. Who knows with cannabis? It may require treatment (e.g. with colchicine) to double....

I posted over in the recent findings thread that these genetics are getting worked out in maize. It’s quite possible that researchers with access to CRISPR or the like could intentionally make inducer males right now. It may well be that the mechanisms of pollen are sufficiently conserved across plant species.

Oddly, inducer males will probably seem horrible from a traditional breeding standpoint. They’re likely to produce seeds with low or super low germination. These ‘dud’ males may be the most valuable males anyone ever runs across! Crazy....

 

[Tonygreen]

Anther culture to make the haploid then colchisine to restore viability and make the double hap. I've attempted to make haploids but my aseptic protocol is lacking... if I had money for a good flow hood I would buy one...

Chimera has the skills and tools to make double haps... saw one other guy with a setup he was using to make tetraploids and I tried mentioning he had the perfect set up to make double haps but he ignored me ha.

Could always try irradiated pollen as an inducer if you could get a hold of some. In the us you can find places that will irradiate seed for you. Probably have to imply you were not doing cannabis work tho ha.

 

[Tonygreen]

Hi all, I am an undergraduate assistant researcher in a plant genetics lab. (Full disclaimer: this does not mean I am a geneticist!) Our lab's chief research is phylogeny of brassica genus. My job just so happens to be development of doubled haploid lines of brassica, focusing on brassica in the trianglle of U (i.e nigra, carinata, oleracea, napus, rappa, juncea) these guys are important because they are at model for studying polyploidy in plants. Anyway it's done by selecting buds that are likely to contain microspores (think of them as gamete stem cells!), then they are treated with mercury chloride to clean the buds without damaging the outer layer of the bud. This is done in very aseptic conditions, over a 5 hour protocol, one mold spore or bacterium will ruin everything! after 10 minutes, the HgCl2 is flushed out, I mash up the buds, put them through a refrigerated centrfuge several times to get the microspores to pellet. then I plate them in a liquid media. Then they are "heat-shocked" at 30 C for 24 hrs to arrest meiosis (inhibiting spindle formation I think, don't quote me!) and facilitate haploid doubling, than incubated at 21 C for ~21 days. If I'm lucky, embryos will form, i grow those on a shaker in the dark for a week maybe. I grow them on solid agar media with a cocktail of hormones and MS media until they form a callus (a lumpy plant-tumor thing), and put it on MS with GA3 hormone and they will hopefully grow shoots, that I grow essentially as if it were a clone. What I'm doing is neat, I'm doing it on other species of brassica as well, stuff there aren't even methods papers on yet! Its essentially like "super-selfing," not sure what caveats would exist in a monoecious plant like Cannabis for something like this, but I would like to figure that out! It would dramatically reduce time needed to make true-breeding lines I would think, but I don't want to make baseless claims. Also, you do sometimes get cool mutants! for example, a mutation that caused the leaves on a botrytis (cauliflower) plant to wrap around the petiole like an elm leaf, and possibly a Siamese twin broccoli. here's a paper on this too! https://www.researchgate.net/public...rd_cauliflower_Brassica_oleracea_var_botrytis

Anther culture using pollen is a quick way to make a cannabis haploid. You have the aseptic protocol skills to do it... then use the haploids to make the double hap. 2 steps to pure homozygosity instead of 1 but still.
The beauty of cannabis is we can get a female to create pollen...

Tried multiple times with a shitty home made flow hood... not good enough.

You should totally fucking do this.

Two double haps crossed would make supreme f1s.

 

[Yard dog]

In contrast, if someone finds (and shares) an inducer male, you could pollinate a female and then simply sprout haploid seeds! In many species these actually show spontaneous chromosome doubling, so you have doubled haploids (I like to think of them as S∞) in one step. Who knows with cannabis? It may require treatment (e.g. with colchicine) to double....

I posted over in the recent findings thread that these genetics are getting worked out in maize. It’s quite possible that researchers with access to CRISPR or the like could intentionally make inducer males right now. It may well be that the mechanisms of pollen are sufficiently conserved across plant species.

Oddly, inducer males will probably seem horrible from a traditional breeding standpoint. They’re likely to produce seeds with low or super low germination. These ‘dud’ males may be the most valuable males anyone ever runs across! Crazy....

As far as I'm aware with regard to them using this with corn, they hit the selected mum with the super inducer to get her genes. They then double this up with treatment, the specific gene they termed "not like dad" is what they require for an inducer.

http://emboj.embopress.org/content/36/6/707

 

[CreeperStipule]

In contrast, if someone finds (and shares) an inducer male, you could pollinate a female and then simply sprout haploid seeds! In many species these actually show spontaneous chromosome doubling, so you have doubled haploids (I like to think of them as S∞) in one step. Who knows with cannabis? It may require treatment (e.g. with colchicine) to double....

I posted over in the recent findings thread that these genetics are getting worked out in maize. It’s quite possible that researchers with access to CRISPR or the like could intentionally make inducer males right now. It may well be that the mechanisms of pollen are sufficiently conserved across plant species.

Oddly, inducer males will probably seem horrible from a traditional breeding standpoint. They’re likely to produce seeds with low or super low germination. These ‘dud’ males may be the most valuable males anyone ever runs across! Crazy....

Very interesting, I read this post last night and then when I tried to sleep the haze cross I smoked kicked in big time, odd with haze I sometimes don't know how high I am until I try to sleep and bing mad mad thoughts....
I kept thinking about Dj's semi sterile males (assuming they are males) and how we could use a sterile male (inducer) like cabbage breeders do ( I think they are recessive and monogenic ??) isn't there a way we could use them for reverse breeding? like you'd have the sterile line (A) and the maintained or maintainer line (B)

 

[GMT]

Why aren't haploids weaker? Half the RNA production, half the cell functionality, surely?

 

[CreeperStipule]

Why aren't haploids weaker? Half the RNA production, half the cell functionality, surely?

I read they got DH's via this method in cabbage with a high success rate, though you need to do a bit of work as in a lot of backcrossing -

The pollen sterility which is controlled by cytoplasmic genes is known as cytoplasmic male sterility (CMS). Usually the cytoplasm of zygote comes primarily from the eggs cell and due to this progeny of such male sterile plants would always be male sterile.

"CMS may be transferred easily to a given strain by using that strain as a pollinator (recurrent parent) in the successive generation of backcross programme. After 6-7 backcrosses the nuclear genotype of male sterile line would be almost identicle to that of the recurrent pollinator strain. The male sterile line is maintained by crossing it with pollinator strain used as a recurrent parent in backcross, since the nuclear genotype of the pollinator is identicle with that of the new male sterile line. Such a male fertile line is known as maintainer line or ‘B’ line and ‘male sterile line is also known as ‘A ‘ line. Cytoplasmic male sterile is not influenced by environmental factor and it resides in maize in mitochondria."

Male Sterility - agriinfo.in

Male Sterility Male sterility is defined as an absence or non-function of pollen grain in plant or incapability of plants to produce or release functional pollen grains. The use of male sterility in hybrid seed production has a great importance as it eliminate the process of mechanical...

agriinfo.in

I read they got spontaneous DH's on page 9 of this pdf... (I was high at the time though, and this will teach me for smoking a haze type... no sleep for me off them)

I would def need another few reads to get my head round it (as a lot went straight over).......

 

[GMT]

Yeah double haploids I get, for cell function it may as well be a standard diploid. But while in the haploid state, surely there has to be observable costs for the plant. If it fails to become a double haploid, I'm assuming it becomes a casualty of science pretty quickly.
I can't really see a cost benefit to the science. If you want a duplicate, take a clone. Where is this useful and cost effective?

 

[CreeperStipule]

it def isn't cost effective at the moment... well it would be useful to turn a heterozygous plant into a homozygous plant for breeding most def.

 

[zif]

Why aren't haploids weaker? Half the RNA production, half the cell functionality, surely?

There are very minor differences between tetraploids and diploids - why wouldn't the same apply to diploids and haploids? I.e., perhaps slightly weaker, but by no means abysmally so....

The haploid state is not really 'half' of anything, after all. It's the state of having one complete genome.

From a traditional breeding standpoint, there is one major concern - haploids only have one copy of each gene, so there's no (obvious) way for them to avoid the effects of any recessive lethal alleles they inherit. Diploid plants are far less likely to suffer from these alleles because they need to get two bad copies (i.e., from their parents, mutations, or both).

On the other hand, evidence from sequencing suggests that, over time, many (important?) genes end up being present as multiple copies, both near their primary transcription site, and, oddly, at random points throughout the genome. It's conceivable that this redundancy is, in part, a hedge against the rare haploid offspring having virtually no chance of surviving.

 

[zif]

Yeah double haploids I get, for cell function it may as well be a standard diploid. But while in the haploid state, surely there has to be observable costs for the plant. If it fails to become a double haploid, I'm assuming it becomes a casualty of science pretty quickly.
I can't really see a cost benefit to the science. If you want a duplicate, take a clone. Where is this useful and cost effective?

The advantage is skipping several years of work developing homozygous parental plants for subsequent hybridization. It's also useful in that the expected weaknesses among the haploid and double haploid plants are very good for eliminating lethal and/or very poorly performing recessive traits from the resulting generation(s). I.e., we *know* that when H/DH plants reach maturity, they have only functional alleles!

it def isn't cost effective at the moment... well it would be useful to turn a heterozygous plant into a homozygous plant for breeding most def.

The overhead of running the kit necessary to produce doubled haploids surely pales in comparison to that required when running the kinds of numbers necessary to produce comparable results from line breeding. It's highly doubtful that 'king corn' would be using DHs if this wasn't the case.

 

[GMT]

Science is great I get it. I just don't need this. Nor can I see anyone who does. Nor would I want either a haploid or double hap plant. For breeding or smoking. There are certain basics being ignored here. Just because it's a haploid, doesn't mean all of its genome is desirable. You're still at the point of blind luck as to what's in it. Only it's weaker. Now if you had identified every gene, it's function, location and desired frequency, and were printing DNA, inserting your designer DNA, then it would be worth it. But when you're playing pin the tail on the donkey, why put the donkey on the other side of town?

 

[CreeperStipule]

Science is great I get it. I just don't need this. Nor can I see anyone who does. Nor would I want either a haploid or double hap plant. For breeding or smoking. There are certain basics being ignored here. Just because it's a haploid, doesn't mean all of its genome is desirable. You're still at the point of blind luck as to what's in it. Only it's weaker. Now if you had identified every gene, it's function, location and desired frequency, and were printing DNA, inserting your designer DNA, then it would be worth it. But when you're playing pin the tail on the donkey, why put the donkey on the other side of town?

Whilst you don't need this, the industry does.. it's set to grow to what something like 90 bill usd in a few years, so fully agree with zif actually that although to me the cost is unattainable it won't be for the players in this, and yes when you compare this to "traditional" breeding it shits on it to be frank, ie a game changer in breeding.
You're playing pin the donkey right now with crosses, how long do you think OG on both sides lasts? we seek homozygous plants plain and simple.
Progression doesn't stop it moves forward and this is a step forward in a big way, what other way do you know of to get hetero to homo and therefore further hybrids?
Going back to haploids being weaker etc, yes smaller plants etc but selection will be better as zif says no masking therefore evolutionary rates will be sped up in the haploid, I'm sure adaption is quicker in a haploid too therefore I'd state a fitness advantage and if any new gene origination is brought about as a benefit (mutation etc) well if these were to be fixed in a traditional way like with a big population they won't be yet they easily could be with the haploid state.

 

[GMT]

While I accept that niche evolutionary forces would have a greater impact on survivor numbers, this doesn't translate into shorter adaptation periods. In order to adapt, the organisms must already posses the gene combos necessary. With the greater availability of genes to select from, a hetro dip, or better yet higher, ploidy count, must provide an increased probability of individual survival. This is the only route to evolution, survival. Remember a field isn't a dojo, too late to learn, live or die. Burbank was a tosser, don't try and teach an old plant new tricks as it were.

A great deal of money has been raised by clever men, telling money men, of the advantages they would gain for just a relatively small amount of money; upfront.

 

[GMT]

Besides, a double haploid line would need to be genetically modified anyway. Beyond what's being discussed. In order to get true breeding, the Y chromosome would need to be added to one of the cells from the chosen double haploid female DNAs. (And the X removed ) and then that new male cell, would have to be grown in Tissue Culture until large enough to survive. Then you would have fairly true breeding, for a while. Not long.

 

[zif]

Science is great I get it. I just don't need this. Nor can I see anyone who does. Nor would I want either a haploid or double hap plant. For breeding or smoking. There are certain basics being ignored here. Just because it's a haploid, doesn't mean all of its genome is desirable. You're still at the point of blind luck as to what's in it. Only it's weaker. Now if you had identified every gene, it's function, location and desired frequency, and were printing DNA, inserting your designer DNA, then it would be worth it. But when you're playing pin the tail on the donkey, why put the donkey on the other side of town?

I disagree re: weakness. Plants that survive as DHs would be stronger than any other homozygous plant you could generate, because the entire genome *must* be weakness-free for that survival to happen.

 

[zif]

Besides, a double haploid line would need to be genetically modified anyway. Beyond what's being discussed. In order to get true breeding, the Y chromosome would need to be added to one of the cells from the chosen double haploid female DNAs. (And the X removed ) and then that new male cell, would have to be grown in Tissue Culture until large enough to survive. Then you would have fairly true breeding, for a while. Not long.

DHs are not made to develop true breeding lines. They are used as parents for F1-hybrids.

 

[GMT]

Several points: at the haploid stage, they would definitely be weaker.
At the double haploid stage, I said I accept they work as diploids, but still have less adaptability due to fewer gene choices.

The only reason to make DH P1 plants is to produce....., Oh shit you're right, the whole purpose is to only produce one generation of plants. What a fuckin pointless exercise. Then what lol.

I was thinking that the line would be created in order to be true breeding for all traits for a few gens. I was thinking that these seeds would then be sold as an ingredient for others to create their own recipe. But no, they're trying to make conformist weed. What a total waste of time. It leads nowhere. Completely useless tech.