pharmacoping
Active Member
ry.
54
8. The THC Pathway
The terpene pathway is important to understand both because it serves as a model for theother biosynthesis reactions, such as the THC pathway, and because the terpene geranyldiphosphate is needed in THC biosynthesis. Similar reactions, albeit at different rates andlocations, occur within plant cells that result in production of THC. The chemicalstructure of THC was first determined in the 1930s (Pertwee, 2006). Knowing thecomplete pathway to its production is considered an important piece of
Cannabis
biotechnology.


ShownhereisthemolecularstructureofTHCAandTHCwitharrowspointingtothevariationinthesidegroup.THCAisthecomponentin
Cannabis
plantsanditisnotuntilitisburnedthatTHCisformed.

Interestingly, it is not until THCA is burned that it becomes chemically modified into amore psychoactive form, which is THC (Hazekamp et al., 2005). The burning causes adecarboxylation reaction, or a loss of a carbon group that is on the THCA molecule,thereby converting it to the more psychoactive THC molecule.However, the THCA component of
Cannabis
is the precursor of THC, so its formationand accumulation within the plant influences the amount of THC when the plant issmoked. Again, part of the THCA molecule is derived from the terpene geranyl
55
diphosphate. Synthesis of THCA begins when a molecule of geranyl diphosphate (amonoterpene) is joined to a phenolic ring (a circular molecule with six-carbons). This iswhy THC is sometimes referred to as a terpenophenolic. Because it has a few extramolecular attachments, the phenolic ring is called olivetolic acid and it is through theenzyme geranylpyrophosphate
livetolate geranyltransferase that forms cannabigerolicacid, or CBGA. The final product after CBGA formation is THCA by way of tetrahydrocannabinolic acid (TCHA) synthase. Subsequently, high levels TCHA arefound in
Cannabis
trichome cavity (Sirikantaramas et al., 2005).
 Alookinsidethe
Cannabis
cell,showinggeranyldiphosphateandolivetolicacidcombiningtoyieldTHCA.
The pathway leading to olivetolic acid is most likely synthesized from three molecules of hexanoyl-CoA. However, work remains to be done to in order to understand the synthesisof THCA in its fullest extent. Details on each enzymatic reaction, their substrates andtheir products have been recently provided (Taura et al., 2007).With all this biochemistry comes the curiosity of why
Cannabis
has evolved to produceTHC-like molecules. It has been hypothesized that the molecules can act as a sunscreenfor the plant (Lydon et al., 1987). In fact, research has shown that THC can absorb UVlight, thus the plants are protected from harmful radiation. Additionally, THC precursorshave believed to have antimicrobial activities, therefore these cannabinoids may also playa role in plant defense.Since the part of the biochemical pathway of THC has been elucidated, picking some of the genes from the pathway for transgenic manipulation is possible. For example, if
56
THCA synthase is attached to the CAMV35S promoter it will be highly over expressed.This would produce transgenic lines of
Cannabis
that are loaded with THCA.Putting these genes into other plants may serve useful to people in countries where
Cannabis
cultivation is illegal. One species of plant that might be desirable to geneticallymodify with THC genes is the weed species,
Amaranthus retroflexus
. This plant is in thefamily Amaranthaceae, also known as the pigweed family. The common name for this plant is redroot pigweed and is consumed as a food in some parts of the world (Kong etal., 2009).One reason for its candidacy for genetic modification stems from the fact that it is aweed; it grows along railroad tracks, in ditches, and even between cracks in the middle of parking lots. Therefore, very little labor would be required from the cultivator to maintainhealthy pigweed plants.A second reason for its candidacy is that the flowers of pigweed are large and bulky. Thiswould provide the obvious advantage of producing large quantities of finished product.Additionally, it needs little water, grows rapidly, produces lots of seed, and tolerates poor soil and harsh growing conditions. In many respects it behaves like
Cannabis
, but islegal. Growing a few plants of pigweed would not send the police to your house. For instance, growing pigweed next to your tomato plants in your garden would not seem thatstrange. Neighbors would not give the situation a second thought.

57
Amaranthusretroflexus
,acandidateforgeneticmodificationwiththeTHCAsynthasegene.Thetopleftcornershowsanup-closeviewofthelargeflowerclustersofthisplant.
58
The prospect of growing a legal THC-containing plant might also seem alluring tomedical marijuana users. Within the US, medical marijuana is currently legal in only ahandful of states. While other countries have legalized or promoted the use of medical
Cannabis
, the US Food and Drug Administration (FDA) has historically declaredmarijuana to have only limited medical potential. This is contrary to continuing scientificfindings and the fact remains many patients currently use medicinal marijuana with or without a doctor's recommendation.The inflorescence (flower) of pigweed can be much larger and bulkier than marijuana,which would allow for production of large amounts of medication for medical marijuana patients. The biotechnology for producing transformed, THC-containing plants might bean effective way to bypass legal issues and still allow sufferers of chronic illnesses toself-medicate. Since
Amaranthus
is known to harbor terpenoid biosynthetic pathways,inserting the THCA synthase gene should result in THC production.Transforming a plant with one gene is relatively straightforward. Inserting multiplegenes, called gene stacking, has proven to be more difficult. In the past researchers had todo laborious transformations starting with one gene, then grow the plant into an adult,and breed it for multiple generations. Only then could they use this stem tissue for creating calluses and insert a second gene. Success was far and few between. Fortunately,many new vector systems, mainly in the form of plasmids, have shown to be moreversatile in their capacity to deliver multiple genes simultaneously (Dafny-Yelin andTzfira, 2007). The emergence of artificial plant chromosomes has allowed putting severalgenes together and inserting them into a vector. With time, the complete THC pathwaywill undoubtedly be inserted into other plant species.
59
9. Smoking Roses and Other Proposals
There are limitless ways in which
Cannabis
and biotechnology will influence oneanother. Having a basic knowledge of science and biology is imperative, but having animagination might prove equally as important. However, thinking of concepts andapplying logical ideas to them begins with a solid science education. This allows one togather reasonable arguments as to possibilities of
Cannabis
transformation that may arisein the near future.Work has already begun with yeast cells (Taura et al., 2007). These small fungi weregenetically modified to express the THCA synthase gene. Workers from the same labwere also responsible for transforming tobacco, albeit under special conditions(Sirikantaramas et al., 2004).
For example, the THCA synthase enzyme had to be provided with the THCA precursor molecule (cannabigerolic acid). The tobacco cellswere also grown in vitro. Nevertheless, the gene for THCA synthesis has been shown tohave the ability to successfully transfer and expressed in organisms other than
Cannabis
.Some of the fastest advances in improving
Cannabis
and other plants have been throughapplication of chemicals or hormones. For example, inducing chromosomal duplicationsin plants has been occurring since the discovery of colchicine. This chemical interfereswith the proteins that pull chromosomes apart during cell division. Applying colchicinehas been shown to cause complete genome duplications. Sometimes this leads todoubling of all gene products and not just the genes. It follows, then, that a
Cannabis
plant treated with colchicine might result in production of twice as much THC than anuntreated plant.Although colchicine is commercially available, performing more drastic geneticexperiments are not so easily available. These require special aseptic conditions andaccess to the necessary technology. Once these obstacles are overcome, transforming
Cannabis
with any gene is simply a game of experimentation.It is indeed possible to control genes and cause them to be upregulated in order toincrease their gene product. To do this, the known gene has to be attached, or ligated, to aspecial region that communicates this to the
Cannabis
cell. This region is called a promoter region, since it promotes the expression of that gene. The promoter region sits just ahead of the gene along the chromosome.Some promoter regions have been found to have such strong expression activity, that theyare routinely used in plant biotechnology. One such promoter is called the CaMV 35S promoter (Venter, 2007). This promoter was first found in a virus, then carefullyremoved, and finally ligated to a plant gene. When researchers did this they found thatwhatever gene was attached resulted in a constant expression of that gene. The CaMV35S promoter has since proven to be a useful promoter to make transgenic plants thatexpress large amounts of a foreign gene.Since there is overlap of the THC and terpene biosynthetic pathways, adding an
60
additional two or three terpene genes to
Cannabis
will likely result in that terpene product. For example, many fruit scents and flavors are terpenes. Most anyone is familiar with the citrus smell of an orange, grapefruit or lemon. This smell is the result of aterpene known as limonene.The biosynthesis of limonene is so well understood that there are multiple transgenic plants that have been made expressing limonene. Putting the limonene gene into
Cannabis
would give the buds a citrus-like smell. While some may find this aestheticallyappealing, others might simply enjoy something different. From a practical standpoint,the paranoia of indoor growers might decrease upon learning that the smell their neighbors are complaining about is lemons rather than from marijuana cultivation.Since the precursor molecules needed early in the pathway of THC are known, increasingthese initial pathway substrates might result in more THC production. IPP and DMAPPare the starting materials for terpenes. Upregulating the genes (isopentenyl diphosphatesynthase and dimethylallyl diphosphate synthase) would provide this possibility. Thesegene sequences are known in other plants, therefore a model for isolation andamplification of the
Cannabis
IPP and DMAPP synthase genes is available.Another interesting experiment focuses around
Cannabis
flowers. Many roses arecurrently sold as so called, double roses. This is because they have two whorls of petals,not just one, as in typical roses. This was brought about not by genetic modification, butthrough discovery of a mutant double flowered rose. The mutant was subsequently bredwith other roses to distribute the mutation through the offspring. Selection for doubleroses and crossing between double roses produced only double roses, so much in fact,that there are complete genetic lines of double flowered roses.One of the most prominent desires from
Cannabis
growers is to increase yield. Manycultivators would rather grow one plant that yields 2 kilos than to grow five or six plantsthat produced this same amount. Luckily for
Cannabis
growers, a single gene controlsflower size, at least in some plant species. Upregulating this gene then, would be of hugeimportance to the
Cannabis
community.A different approach to making larger flowers in
Cannabis
would be to express the genefor petals. The transcription factors of the ABC flowering model could be exploited tofacilitate this goal. Although
Cannabis
lacks petals, manipulation of the ABCtranscription factors could overcome this barrier.Conversely, ignoring the petals and focusing on the sepals could produce a similar outcome. Luckily enough, the A transcription factor controls both sepal and petal production. Therefore, up-regulating the A transcription factor would likely result in budswith enlarged petals and sepals. Ultimately, different experiments would be required tofind the best combination of which genes to up-regulate. In addition to larger buds, producing many more buds seems just as important.
61
Perhaps the goal should not be to make larger flowers or have more of them. Consideringhow plants make their food might equally result in an increase in growth of its buds or atleast the time needed. For example, if the genes for photosynthesis are upregulated,conferring hyper-photosynthetic ability, may shorten the time needed to grow
Cannabis
in the vegetative stage.
Cannabis
producers could have the vegetative state of
Cannabis
finish in two months instead of four months.The possibility also exists that one can manipulate the genetic expression of trichomes.The gene for trichome production has been found and described in detail. With trial anderror, a
Cannabis
plant with twice as many trichomes might result in twice as much THC.Alternatively, the entire
Cannabis
plant can be discarded. Inserting THC-synthesizinggenes into any plant that can be cultured in vitro is a possibility. Roses with THC- producing flowers may soon be available to everyday gardeners. The benefits would beobvious. Since roses are perennials, their flowers can be harvested every year, sometimesmore than one time a year. Roses also have the unique characteristic of being able to bloom multiple times in a season, which would provide a continuous supply of TCH-containing flowers.Before
Cannabis
consumers celebrate these transgenic advances with too muchexcitement, there remains a caveat. If marijuana seed companies choose, they might use amethod similar to that which the agricultural biotech seed companies have chosen. For example, in some transgenic food crops a suicide gene is inserted into the seed so the person harvesting the crop will be unable to use seed from that crop for planting thefollowing year. The suicide gene essentially renders the seed infertile. This was themethod that the large agricultural giant Monsanto used in their terminator technology.If a seed company has invested many months or years developing a plant, they may deemit necessary to protect its secrets and stay in business. For now at least, marijuana seedcompanies appear to be following a different philosophy than that of todays corporateagricultural giants.
54
8. The THC Pathway
The terpene pathway is important to understand both because it serves as a model for theother biosynthesis reactions, such as the THC pathway, and because the terpene geranyldiphosphate is needed in THC biosynthesis. Similar reactions, albeit at different rates andlocations, occur within plant cells that result in production of THC. The chemicalstructure of THC was first determined in the 1930s (Pertwee, 2006). Knowing thecomplete pathway to its production is considered an important piece of
Cannabis
biotechnology.


ShownhereisthemolecularstructureofTHCAandTHCwitharrowspointingtothevariationinthesidegroup.THCAisthecomponentin
Cannabis
plantsanditisnotuntilitisburnedthatTHCisformed.

Interestingly, it is not until THCA is burned that it becomes chemically modified into amore psychoactive form, which is THC (Hazekamp et al., 2005). The burning causes adecarboxylation reaction, or a loss of a carbon group that is on the THCA molecule,thereby converting it to the more psychoactive THC molecule.However, the THCA component of
Cannabis
is the precursor of THC, so its formationand accumulation within the plant influences the amount of THC when the plant issmoked. Again, part of the THCA molecule is derived from the terpene geranyl
55
diphosphate. Synthesis of THCA begins when a molecule of geranyl diphosphate (amonoterpene) is joined to a phenolic ring (a circular molecule with six-carbons). This iswhy THC is sometimes referred to as a terpenophenolic. Because it has a few extramolecular attachments, the phenolic ring is called olivetolic acid and it is through theenzyme geranylpyrophosphate
livetolate geranyltransferase that forms cannabigerolicacid, or CBGA. The final product after CBGA formation is THCA by way of tetrahydrocannabinolic acid (TCHA) synthase. Subsequently, high levels TCHA arefound inCannabis
trichome cavity (Sirikantaramas et al., 2005).
 Alookinsidethe
Cannabis
cell,showinggeranyldiphosphateandolivetolicacidcombiningtoyieldTHCA.
The pathway leading to olivetolic acid is most likely synthesized from three molecules of hexanoyl-CoA. However, work remains to be done to in order to understand the synthesisof THCA in its fullest extent. Details on each enzymatic reaction, their substrates andtheir products have been recently provided (Taura et al., 2007).With all this biochemistry comes the curiosity of why
Cannabis
has evolved to produceTHC-like molecules. It has been hypothesized that the molecules can act as a sunscreenfor the plant (Lydon et al., 1987). In fact, research has shown that THC can absorb UVlight, thus the plants are protected from harmful radiation. Additionally, THC precursorshave believed to have antimicrobial activities, therefore these cannabinoids may also playa role in plant defense.Since the part of the biochemical pathway of THC has been elucidated, picking some of the genes from the pathway for transgenic manipulation is possible. For example, if
56
THCA synthase is attached to the CAMV35S promoter it will be highly over expressed.This would produce transgenic lines of
Cannabis
that are loaded with THCA.Putting these genes into other plants may serve useful to people in countries where
Cannabis
cultivation is illegal. One species of plant that might be desirable to geneticallymodify with THC genes is the weed species,
Amaranthus retroflexus
. This plant is in thefamily Amaranthaceae, also known as the pigweed family. The common name for this plant is redroot pigweed and is consumed as a food in some parts of the world (Kong etal., 2009).One reason for its candidacy for genetic modification stems from the fact that it is aweed; it grows along railroad tracks, in ditches, and even between cracks in the middle of parking lots. Therefore, very little labor would be required from the cultivator to maintainhealthy pigweed plants.A second reason for its candidacy is that the flowers of pigweed are large and bulky. Thiswould provide the obvious advantage of producing large quantities of finished product.Additionally, it needs little water, grows rapidly, produces lots of seed, and tolerates poor soil and harsh growing conditions. In many respects it behaves like
Cannabis
, but islegal. Growing a few plants of pigweed would not send the police to your house. For instance, growing pigweed next to your tomato plants in your garden would not seem thatstrange. Neighbors would not give the situation a second thought.

57
Amaranthusretroflexus
,acandidateforgeneticmodificationwiththeTHCAsynthasegene.Thetopleftcornershowsanup-closeviewofthelargeflowerclustersofthisplant.
58
The prospect of growing a legal THC-containing plant might also seem alluring tomedical marijuana users. Within the US, medical marijuana is currently legal in only ahandful of states. While other countries have legalized or promoted the use of medical
Cannabis
, the US Food and Drug Administration (FDA) has historically declaredmarijuana to have only limited medical potential. This is contrary to continuing scientificfindings and the fact remains many patients currently use medicinal marijuana with or without a doctor's recommendation.The inflorescence (flower) of pigweed can be much larger and bulkier than marijuana,which would allow for production of large amounts of medication for medical marijuana patients. The biotechnology for producing transformed, THC-containing plants might bean effective way to bypass legal issues and still allow sufferers of chronic illnesses toself-medicate. Since
Amaranthus
is known to harbor terpenoid biosynthetic pathways,inserting the THCA synthase gene should result in THC production.Transforming a plant with one gene is relatively straightforward. Inserting multiplegenes, called gene stacking, has proven to be more difficult. In the past researchers had todo laborious transformations starting with one gene, then grow the plant into an adult,and breed it for multiple generations. Only then could they use this stem tissue for creating calluses and insert a second gene. Success was far and few between. Fortunately,many new vector systems, mainly in the form of plasmids, have shown to be moreversatile in their capacity to deliver multiple genes simultaneously (Dafny-Yelin andTzfira, 2007). The emergence of artificial plant chromosomes has allowed putting severalgenes together and inserting them into a vector. With time, the complete THC pathwaywill undoubtedly be inserted into other plant species.
59
9. Smoking Roses and Other Proposals
There are limitless ways in which
Cannabis
and biotechnology will influence oneanother. Having a basic knowledge of science and biology is imperative, but having animagination might prove equally as important. However, thinking of concepts andapplying logical ideas to them begins with a solid science education. This allows one togather reasonable arguments as to possibilities of
Cannabis
transformation that may arisein the near future.Work has already begun with yeast cells (Taura et al., 2007). These small fungi weregenetically modified to express the THCA synthase gene. Workers from the same labwere also responsible for transforming tobacco, albeit under special conditions(Sirikantaramas et al., 2004).
For example, the THCA synthase enzyme had to be provided with the THCA precursor molecule (cannabigerolic acid). The tobacco cellswere also grown in vitro. Nevertheless, the gene for THCA synthesis has been shown tohave the ability to successfully transfer and expressed in organisms other than
Cannabis
.Some of the fastest advances in improving
Cannabis
and other plants have been throughapplication of chemicals or hormones. For example, inducing chromosomal duplicationsin plants has been occurring since the discovery of colchicine. This chemical interfereswith the proteins that pull chromosomes apart during cell division. Applying colchicinehas been shown to cause complete genome duplications. Sometimes this leads todoubling of all gene products and not just the genes. It follows, then, that a
Cannabis
plant treated with colchicine might result in production of twice as much THC than anuntreated plant.Although colchicine is commercially available, performing more drastic geneticexperiments are not so easily available. These require special aseptic conditions andaccess to the necessary technology. Once these obstacles are overcome, transforming
Cannabis
with any gene is simply a game of experimentation.It is indeed possible to control genes and cause them to be upregulated in order toincrease their gene product. To do this, the known gene has to be attached, or ligated, to aspecial region that communicates this to the
Cannabis
cell. This region is called a promoter region, since it promotes the expression of that gene. The promoter region sits just ahead of the gene along the chromosome.Some promoter regions have been found to have such strong expression activity, that theyare routinely used in plant biotechnology. One such promoter is called the CaMV 35S promoter (Venter, 2007). This promoter was first found in a virus, then carefullyremoved, and finally ligated to a plant gene. When researchers did this they found thatwhatever gene was attached resulted in a constant expression of that gene. The CaMV35S promoter has since proven to be a useful promoter to make transgenic plants thatexpress large amounts of a foreign gene.Since there is overlap of the THC and terpene biosynthetic pathways, adding an
60
additional two or three terpene genes to
Cannabis
will likely result in that terpene product. For example, many fruit scents and flavors are terpenes. Most anyone is familiar with the citrus smell of an orange, grapefruit or lemon. This smell is the result of aterpene known as limonene.The biosynthesis of limonene is so well understood that there are multiple transgenic plants that have been made expressing limonene. Putting the limonene gene into
Cannabis
would give the buds a citrus-like smell. While some may find this aestheticallyappealing, others might simply enjoy something different. From a practical standpoint,the paranoia of indoor growers might decrease upon learning that the smell their neighbors are complaining about is lemons rather than from marijuana cultivation.Since the precursor molecules needed early in the pathway of THC are known, increasingthese initial pathway substrates might result in more THC production. IPP and DMAPPare the starting materials for terpenes. Upregulating the genes (isopentenyl diphosphatesynthase and dimethylallyl diphosphate synthase) would provide this possibility. Thesegene sequences are known in other plants, therefore a model for isolation andamplification of the
Cannabis
IPP and DMAPP synthase genes is available.Another interesting experiment focuses around
Cannabis
flowers. Many roses arecurrently sold as so called, double roses. This is because they have two whorls of petals,not just one, as in typical roses. This was brought about not by genetic modification, butthrough discovery of a mutant double flowered rose. The mutant was subsequently bredwith other roses to distribute the mutation through the offspring. Selection for doubleroses and crossing between double roses produced only double roses, so much in fact,that there are complete genetic lines of double flowered roses.One of the most prominent desires from
Cannabis
growers is to increase yield. Manycultivators would rather grow one plant that yields 2 kilos than to grow five or six plantsthat produced this same amount. Luckily for
Cannabis
growers, a single gene controlsflower size, at least in some plant species. Upregulating this gene then, would be of hugeimportance to the
Cannabis
community.A different approach to making larger flowers in
Cannabis
would be to express the genefor petals. The transcription factors of the ABC flowering model could be exploited tofacilitate this goal. Although
Cannabis
lacks petals, manipulation of the ABCtranscription factors could overcome this barrier.Conversely, ignoring the petals and focusing on the sepals could produce a similar outcome. Luckily enough, the A transcription factor controls both sepal and petal production. Therefore, up-regulating the A transcription factor would likely result in budswith enlarged petals and sepals. Ultimately, different experiments would be required tofind the best combination of which genes to up-regulate. In addition to larger buds, producing many more buds seems just as important.
61
Perhaps the goal should not be to make larger flowers or have more of them. Consideringhow plants make their food might equally result in an increase in growth of its buds or atleast the time needed. For example, if the genes for photosynthesis are upregulated,conferring hyper-photosynthetic ability, may shorten the time needed to grow
Cannabis
in the vegetative stage.
Cannabis
producers could have the vegetative state of
Cannabis
finish in two months instead of four months.The possibility also exists that one can manipulate the genetic expression of trichomes.The gene for trichome production has been found and described in detail. With trial anderror, a
Cannabis
plant with twice as many trichomes might result in twice as much THC.Alternatively, the entire
Cannabis
plant can be discarded. Inserting THC-synthesizinggenes into any plant that can be cultured in vitro is a possibility. Roses with THC- producing flowers may soon be available to everyday gardeners. The benefits would beobvious. Since roses are perennials, their flowers can be harvested every year, sometimesmore than one time a year. Roses also have the unique characteristic of being able to bloom multiple times in a season, which would provide a continuous supply of TCH-containing flowers.Before
Cannabis
consumers celebrate these transgenic advances with too muchexcitement, there remains a caveat. If marijuana seed companies choose, they might use amethod similar to that which the agricultural biotech seed companies have chosen. For example, in some transgenic food crops a suicide gene is inserted into the seed so the person harvesting the crop will be unable to use seed from that crop for planting thefollowing year. The suicide gene essentially renders the seed infertile. This was themethod that the large agricultural giant Monsanto used in their terminator technology.If a seed company has invested many months or years developing a plant, they may deemit necessary to protect its secrets and stay in business. For now at least, marijuana seedcompanies appear to be following a different philosophy than that of todays corporateagricultural giants.
