Tetraploid and triploid seeds

[Jozsibacsi32]

Hi, my plan is generating triploid seeds from normal diploid and generated tetraploid plants. Already I have a 4n seedling (DP Desfrán), I hope it will grow normally in the future
Which method is the better: use STS on the 4n plant and pollinate a normal 2n female, or generate female pollen from the 2n plant and pollinate the 4n female? Or both ways are good? Any idea from practice or theoretically?

 

[goingrey]

Take some clones and do it both ways?

Out of interest, how does one identify tetraploidy?

 

[Piff_cat]

I read a really good paper on this. They used a different agent and used it on tissue cultured nodes I think . Bottom line was without high throughput cytography stomatas where the best indicator . Tetraploid stomatas were far larger and less numerous. It's not as easy as looking for 4 nodes. Let me find paper it was cool. The terps were a huge increase. Polyploid leaves had as much terps as diploid buds

 

[Piff_cat]

Here's the article this should help you alot. They take bud explant nodes grow in tissue culture and treat with oryzalin. The paper is awesome I can't believe this is not discussed more!

https://www.frontiersin.org/articles...019.00476/full

 

[goingrey]

Great info Piff_cat, thanks for posting!

So to identify tetraploidy they use some dye and a microscope.

Or in their words:

The ploidy level of the diploid mother plant and in vitro polyploid plants was confirmed by chromosome count. Young healthy roots were harvested from plants and rinsed with tap water to remove all traces of media. The roots were placed in a 1.5 ml microcentrifuge tube with water and pretreated with nitrous oxide for 1 h in a custom-built pressurized chamber at 160 psi to accumulate metaphase cells (Andres and Kuraparthy, 2013). The roots were then fixed in a 3:1 ethanol:acetic acid mixture at room temperature for 24–48 h. The root tips were digested in 1 M HCl for 5 min at 60°C and then rinsed with ice-cold water three times. The root tip cells were then excised and macerated on a microscope slide following the squash method of Tsuchiya and Nakamura (1979) and stained with a drop of 2% acetocarmine. Cells were imaged using a compound microscope (Zeiss Lab A1) with color camera (Zeiss Axiocam 105). Chromosomes were counted in at least three root tip cells per genotype.

 

[Piff_cat]

No problem man happy to help. The 3 observable traits also reliable. 1. Stomata size quantity 2. Pollen grain size 3. Glandular trichome density
not only indicators but desirable traits. Taking it a step further I can see alot of upside in a diploid backcross to triploid. Sterile plants would allow us to greatly increase abiotic stresses leading to higher secondary metabolites production. Without the Possibility of herms and lower potency after accidental fertilization. Also we can increase precursor gpp metabolism which is starting point for cannabinoids and terpenes. This test was done witha type ii plant(1/1 thc/cbd) limited revalency for sativa strains but a good starting point
another important consideration is type of polyploid. This technique strengthens miotic spindles during cell replication leading to double the chromosomes
however the "natural" polyploid is created through un reduced gametes during sexual reproduction. This is a numbers game just matter of popping many seeds and trying to provide conditions conducive to spontaneous whole genome replication. It is through this method which new landraces are created by gene duplication creating novel new functions in new enviorments. I'm no scientist but this is fascinating

 

[djonkoman]

Taking it a step further I can see alot of upside in a diploid backcross to triploid.

do take into account there the triploid is likely sterile.
if you meant backcrossing the tetraploid to a diploid to create a triploid, forget what I said. but crossing anything to the triploid is likely not possible.

as to the benefits, it does sound really interesting from a grower perspective (for breeding lesss so since, even if you have a fertile polyploid like a tetraploid, you can't easily improve it anymore since you can't cross to any diploid variety and get a new fertile line out of it. any line you'd want to cross to the tetraploid you first need to make polyploid too before crossing, so you're giving yourself more work).
however, it might not work out as nicely in practice as theory suggests.
for example, I vaguely remember hearing a comment in a youtube video somewhere pf a breeder who said he'd worked on thois, and the result was that the first steps of seed formation still happened, but it aborted after that. so, you will still get buds full of those white nubby seeds if there is pollen around, you just won't get any full-grown fertile seed. (although, as said, just some random comment I vaguely remember, so it might not be true and do work in practice. but this situation is a real possibility, so you should not count yourself rich based on the theory, without seeing it work out in practice).

imo it's academically interesting to make and study polyploid cannabis, but I doubt whether it will ever really be a thing we'll see used in commercial production.

It is through this method which new landraces are created by gene duplication creating novel new functions in new enviorments.

also a bit of pedantic nitpicking on this statement, you're right in the general idea that spontanious polyploid are likely to have played important roles in evolution, however it would lead to new species, not landraces within the same species (landraces also do not occur wild, by definition they are domesticated and reproduced&sown by humans).
a very cool example imo is how our common domesticated strawberries came to be, which are octoploid, and derive from a natural cross of 2 different wild american (the continents) strawberry species, which (the cross) occurred in a botanic garden in france.

 

[Piff_cat]

thanks for the feedback i am trying to teach myself some science concepts. yes i was referring to creating a triploid crossing a tetraploid to a diploid then selecting a queen to be used as a mother probably taking explants for tissue culture. im interested in using scientific tools to create a diverse gene pool which is metabolically enhanced and using the best results to grow large scale. but im not concentrating so much on population genetics and stabilizing lines for preservation/conservation. i think this is similar to how like fruit trees are bred and used? the landrace comment was definitly over generalized as is the word landrace period. the example im imagining is lets say seeds indigenous to central asia are brought to equatorial africa. from my understanding consistent high temperatures with little change in daylight hours or seasons create a situation conducive to unreduced gametes and whole genome duplication. this double copy then frees up quite a bit of diversity and possibilities which can allow the plants to create novel new uses to thrive in their new home. so for me a method could look something like this
1. take 2 lines which are genetically distant and high heterogenous. pop enough seeds to find some with somatic doubling. cross those to each other and grow out a lot of seeds with certain criteria in mind for selection. take the interesting/outlier/crieria satisfying ladies and decide what to do with them ie line breeding, cross to diploid to make triploid clone, outcross to other interesting/diverse tetraploids youve created on and on. from my understanding there is a mechanism in these strategies which can create a sort of permanent heterosis with the increased diversity. but im a layman so any insight greatly appreciated!

 

[djonkoman]

the example im imagining is lets say seeds indigenous to central asia are brought to equatorial africa. from my understanding consistent high temperatures with little change in daylight hours or seasons create a situation conducive to unreduced gametes and whole genome duplication. this double copy then frees up quite a bit of diversity and possibilities which can allow the plants to create novel new uses to thrive in their new home.

you're not really wrong here on the mechanism, but the scale is a bit wrong. this process acts on a much larger, evolutionary, timescale.
as in, spontanious genome duplications are not that common. but, throughout history they have happened a couple of times, and likely that caused some of the big steps in evolution.

but just ordinary mutation and recombination also create variation that could help plants adapt to new environments. also, a single gene could be duplicated, instead of a whole genome duplication.
so it's not like they need a whole genome duplication to adapt to a new environment.
(also, extra production for all gene products doesn't have to always be good, take for example down syndrome, there also 1 chromosome is copied. through evolution those spontanious polyploids also tend to fall back to diploid, and most of the duplicated genes are lost again)

polyploids are cool, I just think if it comes down to practicalities, my bet is it turns out they're not worth all that effort to make them, also because you're then really limited in crossing in new variation, since all other weedplants are diploid, and thus incompatible.
but I might be wrong, it's certainly a cool project to create polyploids, if you have the tools for it available.

 

[Piff_cat]

I guess I'm not hoping to replicate acclimation . Rather mimic a natural process known to be positive in the wild. Especially for plants with multiple cannabinoid synthases , diverse genotypes or open up lines that are powerful but have had genetic bottlenecks or silenced genes . Like take haze a male. The closest cultivars to pure a are relegated to 4 or 5 f1s salvaged over 30 years. If we take those f1s double em up and tgen open pollinated in one room and do pedigree 1 to 1in other. Playing with these progeny could pull some ancestor throwbacks without outcrossing. It's a niche to start but you can stop the process when your satisfied and use results as clone only or a harvest volunteer for tissue culture. This line bandaid haze ix3.0 breeding strategy reinforces A ancestors and also appears to throw polyploid/aneuploid action. Last study on thc synthase I read found 4 thc synthaae in a haze clone. The Vietnam dalat/viet black also has some funky faciated inflorences roo.

 

[djonkoman]

hmm, the multiple thc-synthases might not be as beneficial as you think.
or it might be.
either way, it's not so clear-cut that multiple copies of thc-synthase=more thc. from what I've read, my interpretation/opinion is that most likely the activity of the thc-synthase is not limiting production, but the amount of available precursor/substrate (CBGa) is more likely the limiting factor I think.
the multiple thc-synthases found in some study could also be explained through either non-functional/low expression copies, or duplicates that just seem very close tp thc-synthase (for example, cbc-synthase was first described as an inactive thc-synthase, before it was discovered it produced cbc instead).

This line bandaid haze ix3.0 breeding strategy reinforces A ancestors and also appears to throw polyploid/aneuploid action.

what do you mean exactly with this? you're not going of off number of branches or fasciation right? because while those phenomenon are sometimes called triploid/polyploid in the weed-community, they're completely unrelated phenomenon, no indication of polyploidy at all.

if you're thinking about recreating an ancestral plant like haze A from an f1 child of it, there is also another way that might get you there more easily.
a process not yet attempted in cannabis afaik, but there are papers about it in arabidopsis.
this process is called reverse breeding. the basic idea is that you can block a specific thing that is needed to create crossovers during meiosis. i.e., you can get reproduction without shufling within the chromosomes, you only get new combinations between the parental chromosomes.
so, from an f1 you can create a collection of homozygous lines, pairs of which can recreate that exact f1 plant, but among those lines will also be an exact copy of the parental plant, but in homozygous form. (i.e. it will give through exactly the same genes to it's progeny as haze A did, but the plant itself won't be exactly the same as haze A, since haze A was not homozygous)

thus, as long as f1's of haze A are still in existence, a copy of haze A could be recreated with this method (ofcourse, it first needs some work to get a working protocol for cannabis, but that's practical details, in theory it should be possible)

 

[Piff_cat]

that is a great idea i have read about that but the way you explained it i got it now. so basically no recombination or crossover? or just significantly less. yea synthase is just like the formula or recipe mostly genetic. but its what the synthase act on im most interested in. geranyl gpp formed melvonate path and gpp serves as the precursor for monoterpenes and cannabinoids. bigger cells organs etc more fuel for the fire. then sequiterpenes use fpp which is a step further past gpp. guess would call it a metabolic hypothesis. similar to using higher temps and nutrients when running co2. increase pre cursor find and find ladies already churning out cannabinoids. also pretty cool in the piffcon results are some clear additive traits for certain terps. guaiol carene sho .03 in f1 .06 for f1 x f1. thats pretty cool and could benefit from polyploid as well. but i really like that breeding backwards man thanks

 

[Piff_cat]

hmm, the multiple thc-synthases might not be as beneficial as you think.
or it might be.
either way, it's not so clear-cut that multiple copies of thc-synthase=more thc. from what I've read, my interpretation/opinion is that most likely the activity of the thc-synthase is not limiting production, but the amount of available precursor/substrate (CBGa) is more likely the limiting factor I think.
the multiple thc-synthases found in some study could also be explained through either non-functional/low expression copies, or duplicates that just seem very close tp thc-synthase (for example, cbc-synthase was first described as an inactive thc-synthase, before it was discovered it produced cbc instead).


what do you mean exactly with this? you're not going of off number of branches or fasciation right? because while those phenomenon are sometimes called triploid/polyploid in the weed-community, they're completely unrelated phenomenon, no indication of polyploidy at all.

if you're thinking about recreating an ancestral plant like haze A from an f1 child of it, there is also another way that might get you there more easily.
a process not yet attempted in cannabis afaik, but there are papers about it in arabidopsis.
this process is called reverse breeding. the basic idea is that you can block a specific thing that is needed to create crossovers during meiosis. i.e., you can get reproduction without shufling within the chromosomes, you only get new combinations between the parental chromosomes.
so, from an f1 you can create a collection of homozygous lines, pairs of which can recreate that exact f1 plant, but among those lines will also be an exact copy of the parental plant, but in homozygous form. (i.e. it will give through exactly the same genes to it's progeny as haze A did, but the plant itself won't be exactly the same as haze A, since haze A was not homozygous)

thus, as long as f1's of haze A are still in existence, a copy of haze A could be recreated with this method (ofcourse, it first needs some work to get a working protocol for cannabis, but that's practical details, in theory it should be possible)

So I have been doing reading on your idea. There are definitely 2 haze a f1 clones still available publicly and another 2 or 3 privately. Man such an exciting idea. It looks like tge main constraint is sterile aneuploid pollen in the first haploid spore step. The number of base chromosomes seems to determine success rate the lower the better. 10 isn't optimal but it looks very doable. I read an article going over different strategies but I'm not scientifically technical enough to pull this off. But if your interested in developing protocol I'd be happy to start sourcing genetics. If not all good any more tips greatly appreciated

 

[Fitzera]

This is all over my head but I thought it would be of interest to mention Breeder Steve is working on monoploidy. Says he is in the process of producing sterile females in 50% of the progeny. He notes that they root in 3-4 days rather than the 10-12 days he sees in the regular females. He also notes that "polyploid tends to make more garbage stenocarpic offspring."
It would seem that his sterile offspring have no pistils..hence no seeds.

This was taken from his IG

 

[djonkoman]

So I have been doing reading on your idea. There are definitely 2 haze a f1 clones still available publicly and another 2 or 3 privately. Man such an exciting idea. It looks like tge main constraint is sterile aneuploid pollen in the first haploid spore step. The number of base chromosomes seems to determine success rate the lower the better. 10 isn't optimal but it looks very doable. I read an article going over different strategies but I'm not scientifically technical enough to pull this off. But if your interested in developing protocol I'd be happy to start sourcing genetics. If not all good any more tips greatly appreciated

the chromosome number thing has to do with how many unique combinations you can get.
for example you have 2 chromosomepairs.
then the f1 plant has 2 chromosomes of parent a (1a and 2a), and 2 from parent b (1b and 2b)
so in this case you could get 4 different haploid combinations:
1a&2a , 1a&2b, 1b&2a and 1b&2b.

with 10 pairs, you have 10^2=100 unique combinations.
so, you need to get 100 viable (not aneuploid or other abnormalities) plants, to have every possible combination.

so if for example 1 in 10 is viable, then you'd need only 40 if you have 2 chromosomepairs, but 1000 if you have 10 chromosome pairs.

It would be cool to develop such a protocol, but unfortunatly I don't have a lab, nor a lot of money. I'm just a student.

 

[gonanchoa]

This is all over my head but I thought it would be of interest to mention Breeder Steve is working on monoploidy. Says he is in the process of producing sterile females in 50% of the progeny. He notes that they root in 3-4 days rather than the 10-12 days he sees in the regular females. He also notes that "polyploid tends to make more garbage stenocarpic offspring."
It would seem that his sterile offspring have no pistils..hence no seeds.

This was taken from his IG

Hi, where can we contact breeder steve?

I bet he learnt much from dj during their time together in Switzerland.

Btw 50% is good but check this patent: https://patents.google.com/patent/USPP27475P2/en

" The particular plant disclosed herein was discovered in the area where the inventor was intentionally cross-pollinating and cultivating plants of cross between ‘Celestial Temple Sativa’ and ‘island sweet skunk’ described above using standard Mendelian breeding procedures well known to those of ordinary skill in the art. This resulted in the F1 generation of the inventor's cross, named ‘Pleadian’. It was in the proximity of plants of the ‘Pleadian’ variety that had become hermaphroditic, in the inventor's garden in Lake Tahoe Calif. that he discovered one female plant that could only be reproduced assexually, by taking cuttings and that plant is the origin of this remarkable new strain. The female plant was discovered in a section of the inventor's hydroponic garden. The plant has been and continues to be assexually reproduced by cutting at the inventor's garden in Lake Tahoe Calif. "

No seeds!! maybe using the equatorian sativa might help?

 

[Piff_cat]

This Is real good info thanks guys. This Ecuadorian sativa sounds like how I believe haze was first created accidentally. A type 4 plant originally from North East Asia ending up in pacific side of south America cross pollinating with drug cannabis approaching South America from the carribean/South african/Indian side. The dif phenos are segregation of founder allele traits in the f2 gen. The type 4 23 ft plant may have been an aneuploid creating a gene dosage imbalance resulting in extreme height. This could also account for sterility. Hollow stems are often found in haze. Also iss has haze in it

 

[gonanchoa]

Haze was a colombian hybrid maintained by the haze brothers. Then it was crossed with several varieties and brought to Amsterdam, check out for sam skunkman. Not all people agree on that history though. Forums has been discussing about it for 3-4 decades already. Oldtimers haze is supposedly a haze inbred from seed who arrived from the US and were mantained by oldtimer. I think he gave out the seeds. Not sure if he is around forums anymore. I think someone did a repro of O.Haze from the 80's given out from sam skunkman. Tom hill haze is also pretty good. Every seedbank has their own version of haze at the end, any good sativa would be termed haze in amsterdam.

Furthermore, there are arguments saying that it was a colombian line that came from seeds imported by lebanese people that emigrated from the lebanese war (thats why you find red phenotypes in haze?). Maybe if some of the colombian lines where hashplants adapted to a milder climate, that is why colombian herb at the 70's was supposedly better than others, it was the first indica-sativa hybrid developed in colombia.

Although its not proven and most is based on personal experience of people who smoke at the time, kept seeds from those imported herbs and started reproducing them at home! Dj took time to describe most of the herbs that were imported at that time to the US. I find it pretty useful, his book has pictures and drawings.

 

[gonanchoa]

This Is real good info thanks guys. This Ecuadorian sativa sounds like how I believe haze was first created accidentally. A type 4 plant originally from North East Asia ending up in pacific side of south America cross pollinating with drug cannabis approaching South America from the carribean/South african/Indian side. The dif phenos are segregation of founder allele traits in the f2 gen. The type 4 23 ft plant may have been an aneuploid creating a gene dosage imbalance resulting in extreme height. This could also account for sterility. Hollow stems are often found in haze. Also iss has haze in it

If you want a variety from manchuria, check out for romulan. Myth says it is a korean reproduced by vietnam vets in the US. There is a guy who did a regular version quite recently. Haven't grown them. There is a chinese research paper that argues cannabis originated there.

 

[Fitzera]

Hi, where can we contact breeder steve?

I bet he learnt much from dj during their time together in Switzerland.

Btw 50% is good but check this patent: https://patents.google.com/patent/USPP27475P2/en

" The particular plant disclosed herein was discovered in the area where the inventor was intentionally cross-pollinating and cultivating plants of cross between ‘Celestial Temple Sativa’ and ‘island sweet skunk’ described above using standard Mendelian breeding procedures well known to those of ordinary skill in the art. This resulted in the F1 generation of the inventor's cross, named ‘Pleadian’. It was in the proximity of plants of the ‘Pleadian’ variety that had become hermaphroditic, in the inventor's garden in Lake Tahoe Calif. that he discovered one female plant that could only be reproduced assexually, by taking cuttings and that plant is the origin of this remarkable new strain. The female plant was discovered in a section of the inventor's hydroponic garden. The plant has been and continues to be assexually reproduced by cutting at the inventor's garden in Lake Tahoe Calif. "

No seeds!! maybe using the equatorian sativa might help?

Morning gonanchoa

Breeder Steve can be contacted via Twitter or Instagram, though he is more active on Twitter I believe. He posted a picture of a pollen patent he is using for the project.

Reading that patent I wonder where he ended up with that strain. From my understanding, this new project he is working on is to allow fields to be sown with seed rather than clones, without the worry of being pollinated whether by males within or hemp/other farms in close proximity.