Interesting Article on Inheritence of Chemical Phenotype

[GMT]

Can I just check on what I believe we are talking about here? They took a sativa and an indica, S1'd a female from both. Then checked the dna markers and potency levels. They then S2'd them. They they crossed the S3 female/male reverted plants to each other. Testing the offspring, they found that 25% leaned towrds the sativa side, the same again to the indica side, and about 50% were middle of the road skunky type plants. Is that fairly accurate?
If so, fair enough, wierd way to do it, but yeah isn't that what we would have expected to happen, albeit with a few hermies to boot?

 

[Grat3fulh3ad]

Can I just check on what I believe we are talking about here? They took a sativa and an indica, S1'd a female from both. Then checked the dna markers and potency levels. They then S2'd them. They they crossed the S3 female/male reverted plants to each other. Testing the offspring, they found that 25% leaned towrds the sativa side, the same again to the indica side, and about 50% were middle of the road skunky type plants. Is that fairly accurate?
If so, fair enough, wierd way to do it, but yeah isn't that what we would have expected to happen, albeit with a few hermies to boot?

Close...
They Took a 'sativa' and an 'indica' and made s1s and tested, made s2s and tested. Then They crossed the s2s to make f1 hybrids, which were consistantly middle of the road type plants. They then Selfed the f1 generation creating an f2 generation which displayed the 25%sat, 25%ind, 50%mix. and the point proved was single locus, or simple mendelian inheritance of the chemical composition of the resins.

 

[GMT]

Ah right, so they made some S1s from some F1s that were a MOTR cross. They got the standard mendalin spread from previously uniform plants. Sorry but what does that prove? This single loci stuff has me confused. Whats the story with that side of it? Where is the important bit that I am missing? If they took a pure ibl sativa, selfed it and got a bunch of indicas, that would be interesting, but, surely genetic inheritance over a few generations is to be expected?

 

[Grat3fulh3ad]

The reason this was done was to try to explain the tripartate differentiation found in nature in regards to this aspect of the plant, and to determine if the CBD and THC and other chemical constituents of the resins were controlled by one genetic factor, or by several different ones.
Alot of the assertions in the article that I found most interesting were tangent to the main point, Such as implying that indicas are actually CBD dom sativas.

 

[GMT]

If it was down to more than one genetic factor, would the same mendalin spread not occur? Assuming that one factor will behave in the same statistical way that another genetic factor will, if 2 were relevant to the observed result, and both resulted in standard mendalin spreads, wouldnt the resluts have been the same standard mendalin spread observed?

 

[GMT]

Aren't the terms sativa and indica nicked from rice breeders. Does sativa mean something specific that is relevant to cannabis? Is it latin or something, or just a label that botanists have applied to cannabis and we use to identify certain characteristics, or phenotypes.

 

[Grat3fulh3ad]

Cannabis Sativa from the latin for useful cannabis...

 

[Grat3fulh3ad]

If it was down to more than one genetic factor, would the same mendalin spread not occur? Assuming that one factor will behave in the same statistical way that another genetic factor will, if 2 were relevant to the observed result, and both resulted in standard mendalin spreads, wouldnt the resluts have been the same standard mendalin spread observed?

Well, here is the long answer, from the article... I have some things I have to go do, But I'll try to break it down later...

It should be acknowledged that these results may also be explained with the hypothesis of two duplicated loci, one encoding for a CBD synthase and the other for a THC synthase, mapping so closely that observation of linkage rupture was impossible in the progenies examined. Such a situation was found in different cases of secondary metabolism genes where duplicated members encoded for enzymes catalyzing either consecutive metabolic steps or alternative reactions from a common precursor. In maize, a family of four duplicated genes (BX2–5) was shown to encode for cytochrome P450-dependent monooxygenases, each catalyzing one of the consecutive steps from indole to 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) synthesis (FREY et al. 1995 ; GLAWISCHNIG et al. 1999 ). In Arabidopsis, genes encoding for different 2-oxoglutarate-dependent dioxygenases (AOP1–3) were identified as responsible for the synthesis of different glucosinolates; these genes map to the same position and code for alternative reactions from a common precursor, leading to 3-hydroxypropyl and 3-butenyl glucosinolate (KLIEBENSTEIN et al. 2001 ). These authors could also identify a recombinant inbred (RI) line endowed with null alleles, accumulating the precursor 4-methylsulfinylbutyl glucosinolate. AOP genes were considered as duplicated, rather than as allelic, essentially on the basis of the presence of a cluster of candidate genes in the sequenced Arabidopsis genome. Besides, little homology was found between AOP and other genes, though a 60–70% sequence homology was found among the cluster components. In the case of cannabinoid genes, however, although the possibility of the presence of duplicated genes cannot be ruled out on the basis of the presented experiments, the consequences of a model with duplicated loci should be examined. Had parental CBD lines carried defective alleles at the THC locus (thc/thc-CBD/CBD) and THC lines at the CBD locus (THC/THC-cbd/cbd), a similar, chemotypically uniform, F1 would have been found, as well as the same segregation of chemotypes in the F2. Theoretically the screening of wide populations should reveal the existence of fixed doubly dominant homozygous (THC/THC-CBD/CBD), showing both cannabinoids: these plants should not segregate on selfing. Such a situation has never been observed during several years of germplasm screening and selfing in the breeding programs. Further consideration suggests that if there were any chance of a cross that separated the two duplicated loci, then the CBG chemotype (thc/thc-cbd/cbd) should be found more frequently than has actually been observed. In fact, in populations where a high frequency of all the three chemotypes is found, as in hashish landraces, the recessive alleles cbd and thc should occur with significant frequency and, because Cannabis of necessity outbreeds, should have a good chance to occur in the homozygous state. Instead, CBG plants have been detected in fiber cultivars that, according to a two-locus model, should have a very high frequency of CBD and thc alleles. Therefore, even extremely low frequencies of the cbd allele should lead to frequent CBG chemotypes, due to the virtual absence of THC alleles in fiber hemp. Conversely, as yet only a few reports of single plants show the CBG chemotype (FOURNIER et al. 1987 ; G. GRASSI and V. G. VIROVETS, personal communications), suggesting a very low frequency for this chemotype, despite the observation that the plants carrying defective alleles suffered no loss of vitality (G. FOURNIER, personal communication).

These facts are more convincingly explained by the rare occurrence of a mutated B0 allele for a defective enzyme at a single locus. HORKAY 1986 estimated the degree of self-fertilization in monoecious populations, like the French cultivars in which the CBG plants were found, at 20–26%, and therefore it is conceivable that a mutated, inactive allele at the locus B could have thrived through repeated and frequent inbreeding until becoming fixed in a few plants.

Although based on negative evidence, it is our opinion that the model of a single allelic locus governing the synthesis of CBD and THC better explains the chemotype distribution in Cannabis populations.

 

[GMT]

Lol, all that to just say,... basically we don't know but we guess that it is so and what we did didn't rule it out......

 

[pipeline]

I've had this study for a while and was about to post it, but I did a search and saw that it was already here. This is essential stuff for plant breeders. Great summaries guys.

Many interesting points in this paper, but one that suprised me was the idea that cannabis has the same magnitude of variation within and between populations.

 

[zamalito]

I've always secretly despised this study . Firstly the cbd/thc ratio is variable in both sativa and indica populations. Most american weedy hemp populations are of the high cbd variety. There's one scientist that closely asociates thcv as the marker cannabinoid for indica (lamarck) and sativa populations that contain thcv have indica in them. Also cbd doesn't cause a sedative high, that's cbn. Cbd is actually a thc blocker and causes the delayed onset long lasting creeper high. In just the right amounts its also responsible for the more energetic and low tolerance building strains. Another possible explanation for the results in the forementioned study is there's possibly a large array of thc/cbd ratio genes. Since they didn't have much representation of the mixed chemotype landrace possibly there's another codominant gene for a 50/50 chemotype. This would explain why 75/25 chemotypes exist and infinite degrees in between.

 

[zamalito]

Daytripper and I had a discussion about this on a thread. I'd provide a link to this however daytripper erased his posts replacing some with insults making me look like a raving lunatic.

 

[Grat3fulh3ad]

I have thought along those lines as well, zamilito... With the incredible amount of variation in the chemical makeup of the resins, a logical conclusion that the composition of the resins could very well be goverened by an array of genes naturally follows...

 

[LookingUp]

Grat3ful, thanks for the link to the article. Interestingly, when I got to the article, I found a link at the bottom of the page to a newer article that references the 2003 paper-
K. W. Hillig and P. G. Mahlberg
A chemotaxonomic analysis of cannabinoid variation in Cannabis (Cannabaceae)
Am. J. Botany, June 1, 2004; 91(6): 966 - 975
When I went to look at that article, I saw right away that they conclude cannabis has 2 species.
I think zamilito makes some good points also.
Here's the link to the Hillig and Mahlberg article:
http://www.amjbot.org/cgi/content/full/91/6/966
-LU

 

[Grat3fulh3ad]

Excellent link! thanks

 

[LookingUp]

Finally got through both articles and had some thoughts. One thing I noticed is that much of the work discussed by de Meijer, et.al. is based on cannabis from hemp strains, although what the collection from the Dutch Pharma company was isn't clear. I also note that although they discuss "pure" chemotypes, in fact all of the plants seems to have at least some THC (and probably CBD - it is the precursor for THC), which seeems to screw up their explanation for the mechanism behind the observed chemotype grouping. I also don't think much of their explaining away problem data with an imaginary inbred mutation showing up in a highly selfed hermaphroditic hemp population - after all, my understanding is that most hemp is grown monoecious (from Clarke). In sum, I don't find a whole lot of useful info from the de Meijer article for my purposes (the development and propogation of high quality, specific medicinal cannabis).

The Hillig article had several interesting parts. They report data based on a fairly wide range of cannabis plants, including both hemp and drug varieties. Their sample does seem to be somewhat restricted from a worldwide geographic sense, but covered the Eastern hemisphere pretty well. Basically, they show about the same thing that Robert Connell Clarke talks about in Marijuana Botany (the appendix on Taxonomy and Nomenclature): the high CBD/low THC chemotype is basically your hemp cannabis grown for fiber; the high THC/low CBD chemotype includes the drug varieties, and the more or less equal TC/CBD chemotype is mostly hemp varieties, but does have a few individuals with higher levels of THC/CBD (I wonder if this isn't more of a chemotype resulting from cannabis domesticated for seed oil?).

The thing I found really interesting in the Hillih & Mahlberg article was their classification of various cannabis strains. It seems like nearly all the strains they classified as sativas had low THC content (hemp origins?), but they had a category they called Narrow Leaf Drug (NLD) Strains which they classified as indicas, but sounded to me like they had what most of us would call sativa characteristics.

I'm going to re-read Clarke, because after looking at these other 2 articles, I'm more convinced than ever that good medicine from cannabis depends on more than just a couple of cannabinoids and their ratios to each other.

Does anyone else think Hillig's NLD indicas sound like sativas, or satica/indica crosses?

By the way Grat3ful , I just put 5 Fourplay seeds into paper towels to germinate 72 hours ago, and moved them to rooters after 36 hours - thought you'd like to see a pic.
-LU

 

[Grat3fulh3ad]

From an article about de Meijer in 1999...

The answer is that Mr Watson and his Amsterdam-based scientists are working to create a stable, plant-based medical product. They want to isolate the beneficial effects of cannabis' various properties and then reproduce them, ad infinitum, from specialised parent plants.

Mr Watson and his Dutch colleague, biochemist Etienne de Meijer, are confident that by using their own exclusive cross-breeding methods, they can develop healthy plants which will combine only the desired chemical make-up of individual medicines.

There will be no generational deterioration and no genetic difference between each plant because they will be bred from themselves: they will be cloned. "You can clone a plant 10 times," explains Mr de Meijer, "and every time it will be exactly the same."

Mr de Meijer has developed his own technique of "self-progeny" - or "selfing" - where he turns half of one female plant temporarily into a male. Fertilising a plant with itself in this way means the same genetic make-up can be reproduced.

"I can make 20,000 clones with 'selfed' parents in two weeks," he says. "Humans may degenerate from inbreeding, but these plants do not. I'm sure I am the first person to apply this method of inbreeding to cannabis and I found the selfing process was amazingly simple."

But the unique research has no market in Holland. "Because the sale of the drug is tolerated in coffee shops, there is no interest - though people don't really know what they are buying," says Mr Watson.

As a result, the seeds that HortaPharm is producing are passed straight on to Britain to take their place in the soil at the ground-breaking facility set up this summer by Dr Geoffrey Guy in south-east England. "We hooked up with Dr Guy in January and right now all we are doing is providing the basic building blocks for his work," says Mr Watson. "We were rather surprised that it would happen in England first."

HortaPharm's sample plants are analysed in the laboratory with a gas chromatographer and with each new batch the team homes in on the plant's distinct chemical components or cannabinoids - THC, CBD, CBC, CBG and THCV. When Dr Guy completes his medical research in Britain, HortaPharm will breed plants to supply the right combination of active ingredients for his treatments. "Once Dr Guy has worked out what he wants in chemical form, we will find him the right physical characteristics, too, by combining desirable features from plants found around the world - high-resin production and resistance to disease," says Mr de Meijer.

HortaPharm is only interested in developing female plants that are sterile, but this is not just to protect their genetic copyright. "If a plant is not kept busy producing seeds, all its energy can go into resin production," says Mr de Miejer.

 

[zamalito]

Is it me or do these scientists seem incredibly naive?

Lookingup there's a few things that are accepted by the scientific community that have changed since clarke's work. Firstly the terms indica and sativa are now a reference to drug and nondrug strains. Indica basically now means varieties high in psychoactive cannabinoids and sativa means low in psychoactive cannabinoids. The other change is the accepted precursur of thc and cbd is now cbg. The bt gene is supposed create and enzyme to convert cbg to thc and the bd gene is supposed to create an enzyme that converts cbg to cbd.

 

[Grat3fulh3ad]

Is it me or do these guys seem incredibly naive?

Lookingup there's a few things that are accepted by the scientific community that have changed since clarke's work. Firstly the terms indica and sativa are now a reference to drug and nondrug strains. Indica basically now means varieties high in psychoactive cannabinoids and sativa means low in psychoactive cannabinoids. The other change is the accepted precursur of thc and cbd is now cbg. The bt gene is supposed create and enzyme to convert cbg to thc and the bd gene is supposed to create an enzyme that converts cbg to cbd.

It's not just you...
I was gonna post up about the chemical precursor issue too, but you've summed it nicely...

 

[zamalito]

The one study "a chemotaxonomic analysis" is the basis for and explains how drug levels determine the species. Basically since thcv and cbv only occur in high thc cannabis and a high thc/cbd ratio only occurrs in <25% of nondrug cannabis it means they're separate species. Nevermind the fact that a high thc/cbd ratio should statistically occur less frequently in high cbd populations.

Pretty lame, huh?

I think since they've found all of these medicinal qualities to cbd and they just want to rationalize keeping high thc plants illegal and make high cbd available by calling them separate species.