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Old 09-09-2020, 04:48 AM #421
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Originally Posted by Sam_Skunkman View Post
Good Luck,
I hope you are correct, mine all had lots of white nubs as well as set dark seeds, did you test them with only Triploid pollen or did you test them with Diploid pollen?
I only tested my 30 different unrelated Triploids (each plant over 6 feet tall 5 copies of each) with Diploid pollen from maybe a dozen unrelated Diploid Males, as that is the problem in the world, do you think that explains the difference? I know that in the world it is Diploid pollen they will run into and set seeds from. Maybe Triploid pollen is just not as viable? We had both THC and industrial hemp Triploids and Diploid Males we used to pollinate, they all set seed and all had even more white nubs than dark set seed. But the dark set seed was 10%-95% of normal yield of seeds, none had zero set seed, not including the white nub seeds found in all the Triploids.
Anyway, I am glad that 3 decades later this work is being pursued again, if it does lead to a useable breeding method, time will tell.
I did trials for 3 years before I gave up. But you know how R&D is most does not work out but you learn what does not work and occassionally do find R&D that moves Cannabis forward. Cannabis R&D is what I loved best besides smoking.
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Hi Sam,

Happy to confirm that these triploids are definitely without seeds. Hsuan and team will be publishing later this fall, but it's very good news. Just cleaned a bucketload of material from different ploidy crossing attempts and there still isn't seed or nubs. Triploids pollinated by tetraploids, triploids, and diploids all yielded no seed. We are still waiting on the results for diploids pollinated by triploids to ensure that the pollen is truly sterile as well.

You guys paved the way on this and I know that our whole team is thankful for the advice and information. I'm grateful to share the same thrill of pursuit that R&D bring and it has been very cool to share this with a pioneer...most days are dead-ends but the good ones make waves that last! Wish you could have made it out this year, hopefully next summer.

Will post links when the formal papers are in press.
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Old 09-09-2020, 06:53 AM #422
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I really do not understand why all of the 30 different Triploid females set seed and all produced white nubbies maybe because we tried the pollination with many diploid males from different backrounds we had hemp males, Thai males, Afghan males, as well as hybrid males that we used. At least one of them did pollinate all the different varieties of 30 Triploid females, I had thought all the diploid males did make viable seeds in the triploids when we did the work. I am curious to see if tetraploid and triploid males pollen are viable to normal diploid females did you also test Tetraploid male pollen? Amazing work, it seems you found what I was looking for?

I agree about R&D most teaches what does not work but when you find something that does work all the R&D is worth it.

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Quote:
Originally Posted by socioecologist View Post
Hi Sam,

Happy to confirm that these triploids are definitely without seeds. Hsuan and team will be publishing later this fall, but it's very good news. Just cleaned a bucketload of material from different ploidy crossing attempts and there still isn't seed or nubs. Triploids pollinated by tetraploids, triploids, and diploids all yielded no seed. We are still waiting on the results for diploids pollinated by triploids to ensure that the pollen is truly sterile as well.

You guys paved the way on this and I know that our whole team is thankful for the advice and information. I'm grateful to share the same thrill of pursuit that R&D bring and it has been very cool to share this with a pioneer...most days are dead-ends but the good ones make waves that last! Wish you could have made it out this year, hopefully next summer.

Will post links when the formal papers are in press.
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Old 09-09-2020, 08:08 AM #423
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Old 09-11-2020, 06:32 PM #424
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Man Sam I was just linked to this thread and I've got say I have the exact same concerns you do. Thank you for adding a voice of concerned interest in these projects from OCBD. Maybe everything is on the up and up but man, an entire catalog of sterile Triploid cultivars impervious to all pollen just doesn't sit well with me. My hope is I'm wrong and we'll never hear anything shady about OCBD but I'd be remiss if I didn't express some concerns and I appreciate your logical skepticism. Hopefully with time either grow reports, phylos testing or further explanations from OCBD will help illuminate the puzzle pieces I'm missing. Interesting project for sure, I'll be follow this whole development closely.

Quote:
Originally Posted by Sam_Skunkman View Post
I really do not understand why all of the 30 different Triploid females set seed and all produced white nubbies maybe because we tried the pollination with many diploid males from different backrounds we had hemp males, Thai males, Afghan males, as well as hybrid males that we used. At least one of them did pollinate all the different varieties of 30 Triploid females, I had thought all the diploid males did make viable seeds in the triploids when we did the work. I am curious to see if tetraploid and triploid males pollen are viable to normal diploid females did you also test Tetraploid male pollen? Amazing work, it seems you found what I was looking for?

I agree about R&D most teaches what does not work but when you find something that does work all the R&D is worth it.

-SamS
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Old 09-14-2020, 04:48 AM #425
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It's weird that they've managed to do what nobody else in cannabis has managed to do, ever. Kinda weird. Not sure if I'm buying it.
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Old 09-19-2020, 05:23 PM #426
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Sam, been thinking about this discrepancy and trying to understand it. How were the initial populations of tetraploids screened (flow cytometry, chromosome squash)? Were the triploids receiving pollen verified in the same way?

What we have seen with tetraploids is that reversion to the diploid state is common after the initial, successful doubling event. We have to take great care on the micropropagation side to prevent this from occurring, and it involves multiple "generations" of culture and/or cutting before relative stabilization. Plants that revert often end up mixoploid; some parts remain 4n while others revert to 2n; if that plant is used for seed or pollen production, some resulting seed would end up 3n, but others would be 2n and receptive to pollen.

One of the fun discoveries summer of 2019 was identifying spontaneous tetraploids in a population grown from seed. A larger question now is at what frequency those events happen (seems often!)--the answer of course will shape how we view the evolutionary history of the species.

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Old 09-19-2020, 06:00 PM #427
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Quote:
Originally Posted by rizraz View Post
Man Sam I was just linked to this thread and I've got say I have the exact same concerns you do. Thank you for adding a voice of concerned interest in these projects from OCBD. Maybe everything is on the up and up but man, an entire catalog of sterile Triploid cultivars impervious to all pollen just doesn't sit well with me. My hope is I'm wrong and we'll never hear anything shady about OCBD but I'd be remiss if I didn't express some concerns and I appreciate your logical skepticism. Hopefully with time either grow reports, phylos testing or further explanations from OCBD will help illuminate the puzzle pieces I'm missing. Interesting project for sure, I'll be follow this whole development closely.
Rest assured that diploid plants are not going away--ever--and that is not one of our goals. Pollen-proof cannabinoid production enables the peaceful coexistence fiber / grain production in areas most hospitable to cannabis production, while also benefitting from "gene stacking" via copy number increase of key genes of agronomic importance (disease resistance, secondary metabolite production, flower timing, etc.). While it's a certainly a big deal, it is not novel; genome doubling is the underlying tool used to make many of our most important crops "more fit" for agriculture.

I chuckled a bit at the mention of using Phylos to verify. A flow cytometer and seedlings is all you need for that. The ploidy of our seed will be independently verified in this way by Oregon State University's Seed Testing Laboratory as part of a standard germination trial.

I am not sure what's up with Phylos at the moment, as they have made no public statements in over a year and Mowgli Holmes has been demoted from CEO to a "strategic advisor". I do know, however, that their SNP-based "galaxy" is a really rudimentary comparison tool and has little to no application in ploidy verification. Their test accounts for ~2000 SNPs. There are many different competing genomics companies offering their own tests, ranging from 15K to 90K SNPS (the latter, Eurofins/Medicinal Genomics, was validated using one of our breeding populations as part of a collaboration with us). We are currently under contract with and testing other SNP platforms with Lighthouse Genomics (Canada) and NRGene (Israel) for novel trait detection.

SNP studies + phenotype data + chemotype data yield markers we can use to accelerate the breeding process for specific traits; however, whole genome data is also necessary to truly understand what is going on "under the hood" and to validate the usefulness of those candidate markers in other populations. We generate all that in house, utilizing PacBio's Sequel II HiFi sequencing and Bionano's optical sequencing to generate WGS for analysis. We have one of the largest--if not the largest--collections of WGS cannabis data in the world, all derived from our breeding projects. In addition to marker validation, these are being fed into a cannabis pangenome comparison tool to identify important structural variants, rates of translocation, and other areas of interest to our breeding team.

The same thing is going on in other crops right now because of the rapid evolution of sequencing technology; we got lucky that these tools became available at the same time "hemp" was legalized in the US. The rate of discovery is mind-blowing to say the least, which makes this thread such a fun piece of US cannabis history...you can see where we started and how it rapidly evolved.
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Old 09-20-2020, 03:30 AM #428
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Thanks for the reply. I removed some things from my post and made them a direct message. Below is just what was leftover so it may read a bit weird as a reply to you.

The white is an interesting project. I think some of the plants used to breed that plant may have been smuggled out of your facility because I was able to source a clone of the white about 4-6 months prior to public release.

Thanks for the replies, glad to hear sterile hemp isn't what you're after. Again this is a position you should clarify in my humble opinion because I think a lot of people (maybe a concerted effort) have been shared information that compares you all to Monsanto and if you're not super educated in Crawford history, the plant or the science, it makes a compelling argument.
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Everything I do is within compliant legal limits for the states in which those projects are conducted in. My opinions aren't always popular, sorry if you're on the wrong side of one. I'm neurodivergent so I'll be making no apologies.

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My current grow dairies

Kali China Breeders Pack plus Bubba Hash and Nepal Jam from Ace Seeds
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BG Cindy from 00, Romulan genetics Rom, Djshorts Temple Flo, plus some extras and clones
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Old 09-20-2020, 03:56 AM #429
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<I am not sure what's up with Phylos at the moment, as they have made no public statements in over a year and Mowgli Holmes has been demoted from CEO to a "strategic advisor".>

He took I think $14 million dollars of investor money, and they want some sort of return. Good luck with that.
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Old 09-20-2020, 03:59 AM #430
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Originally Posted by flylowgethigh View Post
<I am not sure what's up with Phylos at the moment, as they have made no public statements in over a year and Mowgli Holmes has been demoted from CEO to a "strategic advisor".>

He took I think $14 million dollars of investor money, and they want some sort of return. Good luck with that.
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Everything I do is within compliant legal limits for the states in which those projects are conducted in. My opinions aren't always popular, sorry if you're on the wrong side of one. I'm neurodivergent so I'll be making no apologies.

"Nature produces individuals and nothing more" -CHARLES EDWIN BESSEY

My current grow dairies

Kali China Breeders Pack plus Bubba Hash and Nepal Jam from Ace Seeds
https://www.icmag.com/ic/showthread.php?t=374396

BG Cindy from 00, Romulan genetics Rom, Djshorts Temple Flo, plus some extras and clones
https://www.icmag.com/ic/showthread.php?t=374416
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