[oldchuck]

I'm reading that genetic study, The Genetic Structure of Marijuana and Hemp, cited above. My understanding of it is limited but the following strikes me as a good reason to consider Cannabis a single species:

The average FST between hemp and marijuana is 0.156 (S1 Fig), which is similar to the degree of genetic differentiation in humans between Europeans and East Asians [9]. Thus, while cannabis breeding has resulted in a clear genetic differentiation according to use, hemp and marijuana still largely share a common pool of genetic variation.

The genetic difference between Europeans and East Asians does not stike me as sufficient to declare them separate species.

[Only Ornamental]

I understand it in such a way that the nowadays cannabis and hemp varieties are a topsy-turvy of crosses and hybrids between many different 'species'. The seemingly closer relationship of hemp and 'indica' (aka BLD) might be the reason for BLH or broad leaflet hemp commonly associated with southern ecotypes (which BTW are common ancestors of many modern hemp varieties).
One remark: The CAN57 they mention is a Syrian collection designated as Cannabis sp. and NOT as hemp (C. sativa)! Morphologically, it's very similar to an 'indica' hash plant with a stout branched stature, absent stretch, broad leaflets, and the typical 'hash' smell. As a 'wild' collection it might be anything from hemp over multi-purpose variety to pure drug cultivar.

[Sativied]

Originally Posted by Sam_Skunkman

But I want intersex tests, that is what I could use and a lot of other people also.
-SamS

Testlabamsterdam (.nl) claims to offer, translated from their Services page, gender tests to "determine gender and perform a genetic check (M/F/Hermaphrodite)". 100e for 10 samples. Don't know how and if reliable but they've been offering that service for a while already.

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Full pdf: www. docdroid.net/teh6qCm/101007s12229-015-9157-3.pdf.html

For this one:
https://link.springer.com/article/10....229-015-9157-3


The Botanical Review
August 2015
Date: 19 Aug 2015
Evolution and Classification of Cannabis sativa (Marijuana, Hemp) in Relation to Human Utilization
Ernest Small

Abstract
Cannabis sativa has been employed for thousands of years, primarily as a source of a stem fiber (both the plant and the fiber termed “hemp”) and a resinous intoxicant (the plant and its drug preparations commonly termed “marijuana”). Studies of relationships among various groups of domesticated forms of the species and wild-growing plants have led to conflicting evolutionary interpretations and different classifications, including splitting C. sativa into several alleged species. This review examines the evolving ways Cannabis has been used from ancient times to the present, and how human selection has altered the morphology, chemistry, distribution and ecology of domesticated forms by comparison with related wild plants. Special attention is given to classification, since this has been extremely contentious, and is a key to understanding, exploiting and controlling the plant. Differences that have been used to recognize cultivated groups within Cannabis are the results of disruptive selection for characteristics selected by humans. Wild-growing plants, insofar as has been determined, are either escapes from domesticated forms or the results of thousands of years of widespread genetic exchange with domesticated plants, making it impossible to determine if unaltered primeval or ancestral populations still exist. The conflicting approaches to classifying and naming plants with such interacting domesticated and wild forms are examined. It is recommended that Cannabis sativa be recognized as a single species, within which there is a narcotic subspecies with both domesticated and ruderal varieties, and similarly a non-narcotic subspecies with both domesticated and ruderal varieties. An alternative approach consistent with the international code of nomenclature for cultivated plants is proposed, recognizing six groups: two composed of essentially non-narcotic fiber and oilseed cultivars as well as an additional group composed of their hybrids; and two composed of narcotic strains as well as an additional group composed of their hybrids.

[Buckowens]

Sam,
I really like the idea of the intersex tests. But there is one thing that is bugging me about how accurate these tests may be.

I have grown thousands of plants, hundreds of clone only varieties and one thing is always true about the plants, they never act the same. Some varieties that are known for their predisposition for intersex traits, and some that are know for how solid they preform (fewer intersex tendencies), yet every now and again I will get minor male expressions to full blown early male flowering. Or in some cases nothing at all.
How will these tests take into account abiotic or environmental stresses that may cause these expressions?
Not all hermaphrodite plants are hermaphrodite all the time, so what are the perimeters for these tests? If I am screening seeds from a chemdog line or sour diesel line for example, what are the odds that the tests will indicate that all plants are intersex or none of the plants re intersex?
Thanks,
Paco

[Stradel]

Oouu so those wild hemp in my country from 2 to 5 meters long thin leafs and clear optimistic no burnout high when i make milk and coockies from them are indicas muhahahaha this is a sadness laugh because i always think this are sativa caracteristics im so stupid omg all thos years i didnt know the difrence betwen ind and sats i am so confused now.)))

[Donn]

One needs a sense of humor, to be a plant taxonomist.

[Sam_Skunkman]

Here are a few more, new and old. A lot are Molecular Biology, not really my forte. If anyone has links to the full papers post them and I will add the links.
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Purification and characterization of cannabidiolic-acid synthase from Cannabis sativa L. Biochemical analysis of a novel enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid

Futoshi Taura, S Morimoto, Yukihiro Shoyama
Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan.
Journal of Biological Chemistry . 08/1996; 271(29):17411-6. DOI:*10.1074/jbc.271.29.17411

ABSTRACT We identified a unique enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid (CBDA) in Cannabis sativa L. (CBDA strain). The enzyme, named CBDA synthase, was purified to apparent homogeneity by a four-step procedure: ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The active enzyme consists of a single polypeptide with a molecular mass of 74 kDa and a pI of 6.1. The NH2-terminal amino acid sequence of CBDA synthase is similar to that of Delta1-tetrahydrocannabinolic-acid synthase. CBDA synthase does not require coenzymes, molecular oxygen, hydrogen peroxide, and metal ion cofactors for the oxidocyclization reaction. These results indicate that CBDA synthase is neither an oxygenase nor a peroxidase and that the enzymatic cyclization does not proceed via oxygenated intermediates. CBDA synthase catalyzes the formation of CBDA from cannabinerolic acid as well as cannabigerolic acid, although the kcat for the former (0.03 s-1) is lower than that for the latter (0.19 s-1). Therefore, we conclude that CBDA is predominantly biosynthesized from cannabigerolic acid rather than cannabinerolic acid.

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Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity is secreted into the storage cavity of the glandular trichomes.

Supaart Sirikantaramas, Futoshi Taura, Yumi Tanaka, Yu Ishikawa, Satoshi Morimoto, Yukihiro Shoyama
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan.
Plant and Cell Physiology . 10/2005; 46(9):1578-82. DOI:*10.1093/pcp/pci166

ABSTRACT Tetrahydrocannabinolic acid (THCA) synthase is the enzyme responsible for the production of tetrahydrocannabinol (THC), the psychoactive component of marijuana (Cannabis sativa L.). We suggest herein that THCA is biosynthesized in the storage cavity of the glandular trichomes based on the following observations. (i) The exclusive expression of THCA synthase was confirmed in the secretory cells of glandular trichomes by reverse transcription-PCR (RT-PCR) analysis. (ii) THCA synthase activity was detected in the storage cavity content. (iii) Transgenic tobacco expressing THCA synthase fused to green fluorescent protein showed fluorescence in the trichome head corresponding to the storage cavity. These results also showed that secretory cells of the glandular trichomes secrete not only metabolites but also biosynthetic enzyme.


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https://www.jbc.org/content/282/28/20739.full
Identification and Characterization of Cannabinoids That Induce Cell Death through Mitochondrial Permeability Transition in Cannabis Leaf Cells
doi: 10.1074/jbc.M700133200
July 13, 2007 The Journal of Biological Chemistry, 282, 20739-20751.
Satoshi Morimoto 1 , Yumi Tanaka, Kaori Sasaki, Hiroyuki Tanaka, Tomohide Fukamizu, Yoshinari Shoyama, Yukihiro Shoyama and Futoshi Taura

Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan

Cannabinoids are secondary metabolites stored in capitate-sessile glands on leaves of Cannabis sativa. We discovered that cell death is induced in the leaf tissues exposed to cannabinoid resin secreted from the glands, and identified cannabichromenic acid (CBCA) and Δ1-tetrahydrocannabinolic acid (THCA) as unique cell death mediators from the resin. These cannabinoids effectively induced cell death in the leaf cells or suspension-cultured cells of C. sativa, whereas pretreatment with the mitochondrial permeability transition (MPT) inhibitor cyclosporin A suppressed this cell death response. Examinations using isolated mitochondria demonstrated that CBCA and THCA mediate opening of MPT pores without requiring Ca2+ and other cytosolic factors, resulting in high amplitude mitochondrial swelling, release of mitochondrial proteins (cytochrome c and nuclease), and irreversible loss of mitochondrial membrane potential. Therefore, CBCA and THCA are considered to cause serious damage to mitochondria through MPT. The mitochondrial damage was also confirmed by a marked decrease of ATP level in cannabinoid-treated suspension cells. These features are in good accord with those of necrotic cell death, whereas DNA degradation was also observed in cannabinoid-mediated cell death. However, the DNA degradation was catalyzed by nuclease(s) released from mitochondria during MPT, indicating that this reaction was not induced via a caspase-dependent apoptotic pathway. Furthermore, the inhibition of the DNA degradation only slightly blocked the cell death induced by cannabinoids. Based on these results, we conclude that CBCA and THCA have the ability to induce necrotic cell death via mitochondrial dysfunction in the leaf cells of C. sativa.

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Cannabinoids Production by Hairy Root Cultures of Cannabis sativa L.
Sayed Farag, Oliver Kayser*
American Journal of Plant Sciences, 2015, 6, 1874-1884
Technische Biochemie, Fachbereich Bio- und Chemieingenieurwesen, Technische Universität Dortmund, Dortmund, Germany
Abstract:
Tetrahydrocannabinol (THC) derivatives are used clinically as analgesic, anti-inflammatory, appetite stimulant, anti-emetic and anti-tumor cannabinoids. THC and its related compounds are at present obtained by extraction from intact Cannabis plants or chemical synthesis, but plant cell cultures may be an alternative source of production. In the present study, hairy root cultures of C. sativa (Cannabaceae) were induced by incubation of aseptically grown callus culture with solid B5 medium supplemented with 4 mg/l naphthaleneacetic acid in darkness at 25 ̊C. Hairy root growth profiles in shake flask, increased periodically during 35 days of growth cycle. The cannabinoid contents produced in minor levels and remained below 2.0 μg/g dry weight. The contents of can-
nabinoid were analyzed by liquid chromatography and confirmed by mass spectrometry.

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Production of D9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing D9-tetrahydrocannabinolic acid synthase from Cannabis sativa L.
Bastian Zirpel . Felix Stehle . Oliver Kayser
Biotechnol Lett (2015) 37:1869–1875
DOI 10.1007/s10529-015-1853-x
Abstract
Objective The D9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed
intracellularly in different organisms to investigate the potential of a biotechnological production of D9-
tetrahydrocannabinolic acid (THCA) using whole cells. Results Functional expression of THCAS was ob-
tained in Saccharomyces cerevisiae and Pichia (Ko- magataella) pastoris using a signal peptide from the
vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest
volumetric activities obtained were 98 pkat ml-1 (intracellular) and 44 pkat ml-1 (extracellular) after
192 h of cultivation at 15 C using P. pastoris cells. Low solubility of CBGA prevents the THCAS appli-
cation in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid
(CBGA) to THCA. Finally, 1 mM (0.36 g THCA l-1) THCA could be produced by 10.5 gCDW l-1 before
enzyme activity was lost.
Conclusion
Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production

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Molecular analysis of genetic fidelity in Cannabis sativa L. plants grown from synthetic (encapsulated) seeds following in vitro storage
Biotechnology Letters
December 2011, Volume 33, Issue 12, pp 2503-2508
Hemant Lata, Suman Chandra , Natascha Techen, Ikhlas A. Khan, Mahmoud A. ElSohly

Abstract
The increasing utilization of synthetic (encapsulated) seeds for germplasm conservation and propagation necessitates the assessment of genetic stability of conserved propagules following their plantlet conversion. We have assessed the genetic stability of synthetic seeds of Cannabis sativa L. during in vitro multiplication and storage for 6 months at different growth conditions using inter simple sequence repeat (ISSR) DNA fingerprinting. Molecular analysis of randomly selected plants from each batch was conducted using 14 ISSR markers. Of the 14 primers tested, nine produced 40 distinct and reproducible bands. All the ISSR profiles from in vitro stored plants were monomorphic and comparable to the mother plant which confirms the genetic stability among the clones. GC analysis of six major cannabinoids [Δ9-tetrahydrocannabinol, tetrahydrocannabivarin, cannabidiol, cannabichromene, cannabigerol and cannabinol] showed homogeneity in the re-grown clones and the mother plant with insignificant differences in cannabinoids content, thereby confirming the stability of plants derived from synthetic seeds following 6 months storage.

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Recent Advances in Cannabis sativa Research: Biosynthetic Studies and Its Potential in Biotechnology.

Supaart Sirikantaramas, Futoshi Taura, Satoshi Morimoto, Yukihiro Shoyama
Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan.
Current pharmaceutical biotechnology. 09/2007; 8(4):237-43. DOI:*10.2174/138920107781387456
ABSTRACT:
Cannabinoids, consisting of alkylresorcinol and monoterpene groups, are the unique secondary metabolites that are found only in Cannabis sativa. Tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) are well known cannabinoids and their pharmacological properties have been extensively studied. Recently, biosynthetic pathways of these cannabinoids have been successfully established. Several biosynthetic enzymes including geranylpyrophosphate:olivetola te geranyltransferase, tetrahydrocannabinolic acid (THCA) synthase, cannabidiolic acid (CBDA) synthase and cannabichromenic acid (CBCA) synthase have been purified from young rapidly expanding leaves of C. sativa. In addition, molecular cloning, characterization and localization of THCA synthase have been recently reported. THCA and cannabigerolic acid (CBGA), its substrate, were shown to be apoptosis-inducing agents that might play a role in plant defense. Transgenic tobacco hairy roots expressing THCA synthase can produce THCA upon feeding of CBGA. These results open the way for biotechnological production of cannabinoids in the future.

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Production of Δ1-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris

Futoshi Taura, Emi Dono, Supaart Sirikantaramas, Kohji Yoshimura, Yukihiro Shoyama,
Satoshi Morimoto
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Biochemical and Biophysical Research Communications. 10/2007; 361(3):675-80. DOI:*10.1016/j.bbrc.2007.07.079

ABSTRACT Delta(1)-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of Delta(1)-tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted approximately 1.32nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a approximately 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure-function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.
----------------------------------------

[Sam_Skunkman]

I hope for several intersex tests, one for intersex genes and one for stress related intersex expression, I assume they are not the same.
If intersex plants have intersex genes like male and female sex chromsome genes, they can be found, If stress is required to express intersex then the genes that allow intersex expression with stress can be found, I hope.
I am looking, as are others...
-SamS

Originally Posted by Buckowens

Sam,
I really like the idea of the intersex tests. But there is one thing that is bugging me about how accurate these tests may be.

I have grown thousands of plants, hundreds of clone only varieties and one thing is always true about the plants, they never act the same. Some varieties that are known for their predisposition for intersex traits, and some that are know for how solid they preform (fewer intersex tendencies), yet every now and again I will get minor male expressions to full blown early male flowering. Or in some cases nothing at all.
How will these tests take into account abiotic or environmental stresses that may cause these expressions?
Not all hermaphrodite plants are hermaphrodite all the time, so what are the perimeters for these tests? If I am screening seeds from a chemdog line or sour diesel line for example, what are the odds that the tests will indicate that all plants are intersex or none of the plants re intersex?
Thanks,
Paco

[Buckowens]

Originally Posted by Sam_Skunkman

I hope for several intersex tests, one for intersex genes and one for stress related intersex expression, I assume they are not the same.
If intersex plants have intersex genes like male and female sex chromsome genes, they can be found, If stress is required to express intersex then the genes that allow intersex expression with stress can be found, I hope.
I am looking, as are others...
-SamS

Thanks Sam.

[Tonygreen]

Im still going with my theory they all have intersex ability.

I have not seen a plant that won't herm, again my sample size is much smaller than many.
Some have said I have females that wont produce pollen when reversed, but they still produce balls.
Many are more stable than others but if I fuck with em enough they intersex.

Has anyone seen a "pure" female that wont show intersex?