RECENT interesting findings

[Sam_Skunkman]

This was a wild landrace variety I collected 9000 feet in Kashmir, the THC is very low and the seeds sprout very easy, seed production is very easy, seed harvest and collection is very hard as when you touch the plants the slightest the mature seeds seem to be spring loaded and are propelled from the plant. I collected the seeds very carefully with an upside down umbrella with a slit to allow the umbrella under the plant, that worked fairly good.
I suspect they are so easily shattering because they are very wild and man has not bred them or selected them for what man wants, bigger seeds and non shattering seeds in the flowers.
I did grow them at sea level in Santa Cruz to see if the would effect seeds size, it did not they were just as small.
-SamS

 

[Sam_Skunkman]

Well shit, no arrogance intended and apologies to everyone if it came across that way. Thanks for the kind words and redirect if I was out of line. Sam has always provided the best advice in terms of searching for something unique (grow as many plants as possible), so the end of the post was supposed to reflect that sage, but often overlooked wisdom.

I thought Mate Dave was asking about the youPCR picture that Sam posted and how that would / could translate to breeding. We've been working with the platform since last summer. Screening for male / female is robust, accurate, fast, relatively cheap. Screening for active CBDa or THCa alleles is much more difficult. Kevin McKernan @ Medicinal Genomics is always working to make the test better by adding new active allele data as it comes in, but the test is very temperature sensitive and we are still getting occasional false positives on the CBDa side. We use the youPCR screen to segregate type I / type IV and type III / type IV in our current projects.

The platform works great for type I / II / III segregation that many people are working on to breed better type IIs and IIIs. When a pure THC parent (P1) is crossed with a pure CBD parent (P2) and the resulting progeny open pollinated / selfed, the square for F2s looks like this (generated from the previously linked website) :

T = functional THCa allele
t = nonfunctional THCa allele
D = fuctional CBDa allele
d = nonfunctional CBDa allele

View Image
View image in gallery​

Which of the 9 types groups had the highest total Cannabinoids and which group had the highest THC only? CBD? CBG?
I am wondering if the Cannabinoid genes or the genes that allow high expression, lots of big resin heads for example are the most important.
When you say functional THCa allele you are referring to the THC synthase correct?

-SamS

 

[Sam_Skunkman]

Thanks Sam. 12 seeds to the gram??? That's incredible. Our largest is 40 seeds per gram and I have a hard time imagining how it could get any bigger. Very cool.

Day neutral are definitely lower in total cannabinoid content, but not prohibitively so. We have them averaging 12% CBD in our type III field varieties and 10% CBG in our type IVs. Their photoperiod counterparts have about 20% higher content on average (15% and 12%, respectively). (note: all tests performed by NELAP accredited labs).

Two other things to think about when considering the relative efficiency of day neutral vs. photoperiod: (1) stem weight and (2) multiple harvests. We are finding that the average stem weight (as a proportion of total plant weight) on full season photoperiod varieties is about 27%; day neutrals come in at 18%. Autos allow at least two harvests per season in a decent production environment or five if you plan to grow in the central valley of California...

Having plants that can reliably finish in late July / early August allows certain locations (i.e. Oregon's Willamette valley) to field dry; when costly drying infrastructure is removed from the equation and finished plants can be picked up with a combine, day neutrals start to look pretty appealing.

Here's one of our berry flavored type III autos; they have more resin than many photoperiod varietals.

View Image​

Is the plant seeded? Looks like it. I do agree this auto does have resin. Does it finish flowering and frost out more like normal drug Cannabis? That was the problem with Autos 25 years ago they just kept on making flowering biomass and never really stopped making flowers and frosting out. `if you test the DNA of any CBD Autos it would be interesting to see if they contained FINOLA DNA as I suspect, even if not it would be interesting to understand the relationships in the AUTO varieties.

-SamS

 

[socioecologist]

Is the plant seeded? Looks like it. I do agree this auto does have resin. Does it finish flowering and frost out more like normal drug Cannabis? That was the problem with Autos 25 years ago they just kept on making flowering biomass and never really stopped making flowers and frosting out. `if you test the DNA of any CBD Autos it would be interesting to see if they contained FINOLA DNA as I suspect, even if not it would be interesting to understand the relationships in the AUTO varieties.

-SamS

It was indeed seeded; F1 in the picture, now in the F4. Your description of their growth is spot on, though they continue to produce copious resin even while building more and more flowers. Great oil producers with complex astringent berry flavors on this one. I don't know for sure, but I suspect all of the autos on the market today were originally derived from Lowryder. The bigger question is what that particular plant (Lowryder) was derived from. I doubt it is Finola; when I grew the original Lowryder and Lowryder II autos in the mid-2000s, it performed nothing like the Finola we experimented with the last few years.

Can the P1 and P2 of Finola remain in veg state under 24 hours of light? Or am I making an incorrect assumption about Jace's seed making strategy?

One of our ongoing research projects is to locate and better understand the genes affecting flowering in cannabis. Plants like these (autos, Finola, and hybrids) are all helping us get a better handle on what's going on under the hood.

RE: the Punnet square. That was just for reference on F2 genotype segregation for the person asking about it. You are correct that certain genotypes tend to produce higher levels of cannabinoids. Homozygous > Heterozygous for pure types (I, III), with heterozygous type IIs producing the most overall.

 

[zif]

The genetics of haploid inducers in maize are becoming clearer (see below). I wonder how far away an inducer male is for the cannabis community? Or is he already out there somewhere?

If so, I wonder what his effects would look like? Would he cause females to have a normal appearing seed set, but with a very high percentage of those seeds nonviable?

Gilles et al. (2017) Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maize. doi:10.15252/embj.201796603

 

[nattyroots]

they should teach genetics in high school with marijuana as an example. I think students would learn a lot faster.

 

[TychoMonolyth]

they should teach genetics in high school with marijuana as an example. I think students would learn a lot faster.

I'm going way back here. In the mid 70s, a couple friends and I were smoking a joint outside a door in grade 12 and my math teacher came out and caught us. All he did was take it, took a good haul, and handed it back. "don't tell anyone guys. Go around the corner to finish it". He was the only teacher with " long hair". Lol

Great teacher BTW. His classes were fun (for a math class)..

 

[Sam_Skunkman]

http://emboj.embopress.org/content/early/2017/02/22/embj.201796603

-SamS

The genetics of haploid inducers in maize are becoming clearer (see below). I wonder how far away an inducer male is for the cannabis community? Or is he already out there somewhere?

If so, I wonder what his effects would look like? Would he cause females to have a normal appearing seed set, but with a very high percentage of those seeds nonviable?

Gilles et al. (2017) Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maize. doi:10.15252/embj.201796603

 

[bsgospel]

Subcellular localization defines modification and production of Δ9-tetrahydrocannabinolic acid synthase in transiently transformed Nicotiana benthamiana.

https://www.ncbi.nlm.nih.gov/pubmed/29619743

CONCLUSION:
Nicotiana benthamiana is a suitable host for the generation of cannabinoid producing enzymes. To attain whole pathway integration, careful analysis of subcellular localization is necessary.

 

[socioecologist]

CONCLUSION:
Nicotiana benthamiana is a suitable host for the generation of cannabinoid producing enzymes. To attain whole pathway integration, careful analysis of subcellular localization is necessary.

Thanks for sharing this. Insertion of THCa synthase into tobacco = successful. Note that this was not the insertion of the full cannabis chemical generation apparatus, just THCa. To produce THCa, they had to place the altered isolated tobacco plant cells in a CBGa rich substrate (i.e. the plant doesn't make precursors natively).

The additional resulting compounds produced are, to me, the really interesting part of this study. The last paragraph of the paper notes:

"THCAS does convert CBGA to different products in a plant matrix, suggesting that enzyme promiscuity additionally contributes to the diversity of cannabinoids in Cannabis."

That particular THCa synthase, in vitro, produces CBCa as well (albeit 4x less affinity). Know your synthases!

 

[Sam_Skunkman]

Cannabis Systematics at the Levels of Family, Genus, and Species
John M. McPartland

Cannabis and Cannabinoid Research
Volume 3.1, 2018
DOI: 10.1089/can.2018.0039

 

[bsgospel]

So the C. sativa L. at the conclusion. Is the L Linneus? Lamarck?

Great read!

 

[mean mr.mustard]

Linnaeus

 

[socioecologist]

Newest preprint paper from George Weiblen's UM group working together with Sunrise Genetics. This is fascinating and has profound implications for breeders.

Christopher J Grassa, Jonathan P Wenger, Clemon Dabney, Shane G Poplawski, S Timothy Motley, Todd P Michael, C J Schwartz, George D Weiblen. "A complete cannabis chromosome assembly and adaptive admixture for elevated cannabidiol (CBD) content.

https://doi.org/10.1101/458083

Full paper available here:
https://www.biorxiv.org/content/early/2018/11/01/458083.full.pdf+html

 

[Sam_Skunkman]

A physical and genetic map of Cannabis sativa identifies extensive rearrangement at the 1 THC/CBD acid synthase locus

Kaitlin U. Laverty, Jake M. Stout, Mitchell J. Sullivan, Hardik Shah, Navdeep Gill, Larry Holbrook, Gintaras Deikus Robert Sebra Timothy R. HughesJonathan E. Page, and Harm van Bakel

https://genome.cshlp.org/content/early/2018/11/07/gr.242594.118.full.pdf+html

 

[Sam_Skunkman]

https://www.marijuanaventure.com/decoding-cannabis-genetics/

More similar work but their paper is not completed or published.

 

[bsgospel]

"Jonathan E. Page and Jake M. Stout have filed Patent WO2015196275 on the nucleotide sequence encoding the enzyme CBCAS based reagents, and methods for producing cannabinoids and/or altering cannabinoid production."

Lot of distractions/loud room mates at the house now- I can't work this sentence out. Is this a patent on CBCAS or the reagents and method used to get these results....or all resultant reagents that the CBCAS yielded?

Interesting results nonetheless.

Edit: This says it better under Claims

https://patents.google.com/patent/WO2015196275A1/en

 

[bsgospel]

https://www.ncbi.nlm.nih.gov/pubmed/30610611

"Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Hippocampus: Cannabidiol Blunts Δ9-Tetrahydrocannabinol-Induced Cognitive Impairment."

Abstract
At present, clinical interest in the plant-derived cannabinoid compound cannabidiol (CBD) is rising exponentially, since it displays multiple therapeutic properties. In addition, CBD can counteract the undesirable effects of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) that hinder clinical development of cannabis-based therapies. Despite this attention, the mechanisms of CBD action and its interaction with Δ9-THC are still not completely elucidated. Here, by combining in vivo and complementary molecular techniques, we demonstrate for the first time that CBD blunts the Δ9-THC-induced cognitive impairment in an adenosine A2A receptor (A2AR)-dependent manner. Furthermore, we reveal the existence of A2AR and cannabinoid CB1 receptor (CB1R) heteromers at the presynaptic level in CA1 neurons in the hippocampus. Interestingly, our findings support a brain region-dependent A2AR-CB1R functional interplay; indeed, CBD was not capable of modifying motor functions presumably regulated by striatal A2AR/CB1R complexes, nor anxiety responses related to other brain regions. Overall, these data provide new evidence regarding the mechanisms of action of CBD and the nature of A2AR-CB1R interactions in the brain.

 

[Azaghal]

Hi everybody,

just a recent interesting finding :

"Gene Networks Underlying Cannabinoid and Terpenoid Accumulation in Cannabis "

http://www.plantphysiol.org/content/early/2019/05/28/pp.18.01506

and the full PDF article :

http://www.plantphysiol.org/content/plantphysiol/early/2019/05/28/pp.18.01506.full.pdf

And a little bit info on the lead author who also "co-founded an agricultural biotechnology company, Dewey Scientific" :

https://plantae.org/research/r-plan...-plant-physiology-first-authors-jordan-zager/

https://dailyevergreen.com/46669/news/agriculture-business-wants-to-open-cannabis-farm/

Cheers

 

[bsgospel]

"Glandular trichomes are specialized anatomical structures that accumulate secretions with important biological roles in plant-environment interactions. These secretions also have commercial uses in the flavor, fragrance, and pharmaceutical industries. The capitate-stalked glandular trichomes of Cannabis sativa (cannabis), situated on the surfaces of the bracts of the female flowers, are the primary site for the biosynthesis and storage of resins rich in cannabinoids and terpenoids. In this study, we profiled nine commercial cannabis strains with purportedly different attributes, such as taste, color, smell and genetic origin. Glandular trichomes were isolated from each of these strains and cell type-specific transcriptome data sets were acquired. Cannabinoids and terpenoids were quantified in flower buds. Statistical analyses indicated that these data sets enable the high-resolution differentiation of strains by providing complementary information. Integrative analyses revealed a coexpression network of genes involved in the biosynthesis of both cannabinoids and terpenoids from imported precursors. Terpene synthase genes involved in the biosynthesis of the major mono- and sesquiterpenes routinely assayed by cannabis testing laboratories were identified and functionally evaluated. In addition to cloning variants of previously characterized genes, specifically CsTPS14CT ((-)-limonene synthase) and CsTPS15CT (β-myrcene synthase) we functionally evaluated genes that encode enzymes with activities not previously described in cannabis, namely CsTPS18VF and CsTPS19BL (nerolidol/linalool synthases); CsTPS16CC (germacrene B synthase); and CsTPS20CT (hedycaryol synthase). This study lays the groundwork for developing a better understanding of the complex chemistry and biochemistry underlying resin accumulation across commercial cannabis strains."