As Legal Weed Hits Canada, Scientists Race to Study and Improve a Once

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Cannabis samples grow under specialized lights at Anandia Labs in Vancouver.

A GOLD RUSH FOR CANNABIS

onathan Page has been around cannabis Some researchers, such as Page (who still all his life. Growing up on Canada’s As legal weed hits dabbled in cannabis research during his dec- NATURE J Vancouver Island in the 1970s, he was ade at the NRC), are well prepared to take surrounded by hippie beachcombers and Canada, scientists advantage of Canada’s great green rush. But dope smokers. So after earning a PhD in plant botanists of all stripes are now turning to the biology and phytochemistry, he felt completely race to study and plant, for both the funding opportunities and at ease working with the plant Cannabis sativa improve a once- the uncharted science. as a postdoc in Germany in the early 2000s. “You’re talking about a plant that’s a century FOR MACDONALD JAMES During that time, Page helped to characterize forbidden plant. out of date in terms of modern breeding a pair of genes that some varieties of the plant techniques and scientific development,” says uses to make fragrant oils responsible for BY ELIE DOLGIN Ernest Small, a botanist with Agriculture and pine- and lemon-like aromas 1. And during an Agri-Food Canada in Ottawa who has studied interview for a position with Canada’s National other countries, most notably Israel, have cannabis off and on since 1971. Research Council (NRC), Page proposed simi- made a concerted effort to support agricul- lar projects to reveal how cannabis produces tural research into cannabis, full legalization RESEARCH BLUNTED pharmaceutically active compounds known as in Canada has brought with it unparalleled When Page first moved back to Canada 15 years cannabinoids. access to money for basic research on the plant. ago, he initially resigned himself to studying a He got the job, but was dismayed when Most of the country’s 129 licensed cannabis close relative of cannabis, the hop plant Humu- he showed up to start his lab group in 2003 producers are now clamouring to work with lus lupulus, which is used in brewing beer. But at the NRC’s Plant Biotechnology Institute in scientists on everything from gene mapping he doggedly pursued avenues to keep working Saskatoon, Saskatchewan. Page recalls his boss and metabolic engineering to optimal drying on pot. Page secured a licence to grow indus- saying: “You’re not going to work on cannabis techniques and growing practices. And as part trial hemp, a variety of cannabis cultivated for here. We’re the government.” of an effort to corner the global legal cannabis its fibre that produces only trace amounts of tet- What a difference a change in policy makes. market — one that’s conservatively forecast to rahydrocannabinol (THC), the mind-altering On 17 October, Canada became the second top US$57 billion within a decade — federal chemical responsible for cannabis’s high. Even- country in the world, after Uruguay, to legal- and provincial governments in Canada are put- tually, he hooked up with the sole company ize cannabis for all uses. And although a few ting up millions of dollars to support research. contracted at the time by the government to

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of the cannabis industry.” That’s why many companies investing in NATURE research are starting with the fundamentals. “There are these super basic, huge questions that need to be answered,” says Greg Baute, who used to breed tomatoes at Monsanto in Woodland, California, and moved north this

JAMES MACDONALD FOR FOR MACDONALD JAMES year to head breeding and genetics at Anandia’s new research facility in Page’s home town of Comox. “You can do these really straightfor- ward experiments and get these huge results.” JOINT VENTURE In a suburban Toronto mall, nestled alongside a paint store, sits a nondescript brick building, home to TerrAscend, a cannabis producer that has been shipping its product for about a month in anticipation of 17 October. Inside, past a barbed-wire fence and several layers of electronic security, are grow rooms full of Shishkaberry, CBD God Bud and Cold Creek Kush, cultivars valued for their sleep-inducing, Many basic agricultural experiments have yet to be performed with cannabis. antidepressant and stress-relieving properties, respectively. produce the plant for medical purposes. alone, Canadian cannabis companies raked in Nearly all the plants are unpollinated females, Page unpicked the pathway that leads to the close to Can$2 billion (US$1.5 billion) — more called ‘sinsemilla’ (meaning ‘without seed’), formation of THC and cannabidiol (CBD) than half of all the funding raised by legal can- that produce the highly potent flowering tops, — cannabis’s other main medically impor- nabis firms worldwide — and the industry is or buds, rich in THC and other cannabinoids. tant compound2. Together with molecular on track to triple that number in 2018. Males, with their pollen-filled sacs, are not only geneticist Timothy Hughes at the University of Anandia was one of the many beneficiar- superfluous — the female plants are all propa- Toronto, he sequenced the genome of a potent ies. After securing more than Can$13 mil- gated through cuttings — but also avoided for pot variety called Purple Kush3. But, says Page, lion (US$10 million) in private investment, fear of scrambling genes in an uncontrolled way. “the NRC was still totally unsupportive of this the company got snapped up earlier this year Yet, TerrAscend has a small space in the back work”. So, in late 2013, he moved to Vancou- by industry heavyweight Aurora Cannabis in reserved for males. Back in June, the company ver to start a cannabis biotechnology company Edmonton, for Can$115 million. “That is a launched a research and development arm in called Anandia Labs. major gesture of confidence,” says Cam Bat- conjunction with scientists at two Ontario uni- In one of Anandia’s first projects, Page tley, chief corporate officer at Aurora, adding versities who will use the sequestered plants for worked with Sean Myles, a population geneti- that science and innovation are key to growing experiments that are standard in agriculture but cist at Dalhousie University’s Agricultural “a globally competitive company that will be have rarely been done with cannabis. The scien- Campus in Truro, Canada, to genetically built to last”. tists will mate the plants, coax them to produce characterize 124 samples of cannabis4. The The sentiment is a relatively new one, says seeds and then expose the seeds to chemical analysis showed that the commercial labelling Michael Ravensdale, a plant pathologist who mutagens in the hope of finding new desirable of subtypes indica and sativa rarely matched leads production at the firm CannTrust in traits — pest resistance, say, or increased toler- the plants’ DNA profiles. And different sam- Vaughan. “Science was in short supply, but it’s ance to environmental stresses such as drought. ples marketed under the same madcap varietal going to be very important for the next chapter Scientists involved with the TerrAscend name — White Widow, for example — often turned out to have wildly divergent genetics. A SMOKING-HOT SECTOR “This is absolutely unthinkable in any other Business deals in the cannabis industry have spiked in Canada since the nation decided to legalize recreational legitimate agricultural crop,” says Myles. “You use. But most of the capital has gone to companies involved in growing and selling. Canadian companies’ can’t put a Mackintosh apple on the shelf and investments in research and development are catching up to the levels seen elsewhere in the world. pretend it’s a Honeycrisp.” 3.5 Despite garnering publicity for the research, United States Agricultural technology Canada Biotech/pharma Private science Page had trouble pulling in capital. Then came 3.0 and technology the election in October 2015 of Prime Minis- Rest of world Cultivation and retail investments topped 2018 gures are as of 5 October $100 million ter Justin Trudeau, who promised during his 2.5 ADVISORS SOURCE: VIRIDIAN CAPITAL campaign to legalize cannabis. “It changed attitudes almost overnight,” Page says. Although the Natural Sciences and Engi- 2.0 neering Research Council of Canada doesn’t have a dedicated cannabis initiative, the agency 1.5

has funded dozens of projects focused on can- Capital (US$ billion) nabis biology and cultivation. Genome Canada 1.0 and other government-backed organizations have made research funds available, too. More 0.5 substantially, private investment dollars have come pouring into the country’s cannabis 0 industry (see ‘A smoking-hot sector’). Last year 2015 2016 2017 2018

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THE WIDE WORLD OF WEED Legal restrictions haven’t completely stymied cannabis research.

Although cannabis is now legal in some form my working hours,” Vergara says. public and private support. He obtained a in all US states but one, the plant, including Even at the University of Mississippi in licence from the state of New South Wales hemp, is still illegal at the federal level — and Oxford, which is licensed by the federal to grow cannabis, and financing from an all research conducted on cannabis at any government to grow cannabis for health industrial hemp company to chemically university must abide by federal regulations, studies, researchers are operating under characterize a global collection of cannabis or else jeopardize government funds for the restrictive rules. The university can procure varieties13. “We want to understand what the institution. plants only from federally approved vendors, scope is for metabolic engineering,” King Extracted DNA samples are permitted, and so it has no access to the THC-rich says. and the government is gradually becoming varieties commonly found in recreational and But nowhere has been as supportive more permissive of hemp cultivation. But any medicinal cannabis shops. Instead, scientists of cannabis research as Israel. It was here work on the basic biology of cannabis with there are using chemical stimulants to boost that Raphael Mechoulam, an organic higher levels of the psychoactive substance THC levels in moderate-strength plants. “My chemist, isolated THC and cannabidiol and tetrahydrocannabinol (THC) is strictly off- hands are tied,” says Mahmoud ElSohly, who determined their chemical structures in limits, and there’s no funding available for oversees the operation. “I have to work with the 1960s. The country has since sought non-health-related research on the plant. what I have.” to establish itself a world leader in the “Everything we’ve done has been absolutely Even in Uruguay where cannabis is study of medical cannabis. And unlike in shoestring,” says plant biologist George completely legal, hurdles still exist. In 2013, some countries, including Canada, where Weiblen at the University of Minnesota in the country became the first to legalize government scientists still essentially can’t Saint Paul. cannabis for recreational use, but researchers access high-THC varieties of the plant, Israel’s That’s led to a few creative workarounds. have faced bottlenecks in gaining access agriculture ministry last year built a national In Colorado, where cannabis has been legal to the plant, according to an analysis12 in centre for medical-cannabis research. for recreational use since 2014, evolutionary March, and there’s no public money to foster Housed at the Volcani Center in Rishon geneticist Daniela Vergara, at the University research. “I get very, very little funding,” says LeZiyyon, the government’s cannabis farm of Colorado Boulder, created a foundation Astrid Agorio, a plant molecular geneticist at includes thousands of plants spread across to support projects she can’t do on campus. the Clemente Estable Institute of Biological several greenhouses and indoor growing Unlike her university, the foundation can Research in Montevideo who is attempting to facilities. There, plant biologist Nirit Bernstein accept funding from the cannabis industry exhaustively profile the genetic structure of is trying to perfect cultivation and weed — money she then spends on sampling two varieties of Uruguayan cannabis with a out bad practices. “We have to develop DNA from cannabis plants bred at local grant of about US$6,000. science-based protocols for optimizing the dispensaries and growers. Although she can In Australia, where medical use is allowed, cultivation of this magical plant,” Bernstein analyse DNA sequence data at the university, plant geneticist Graham King at Southern says. “There’s very little scientific information “I don’t touch the plant on campus or during Cross University in Lismore has found both that’s available.” E.D.

spin-off, including plant geneticists Peter count. At first blush, says Hepworth, “the plants should have optimal properties instead of McCourt and Shelley Lumba at the University are definitely bigger”. But the scientists still need waiting months for the plants to mature. of Toronto, plan to mutagenize six varieties of to finish their analyses to determine whether Only a small number of markers exist for cannabis with the aim of obtaining improved the ‘tetraploid’ cannabis lines have elevated cannabis, however, in large part because few versions of some of the company’s go-to stock. levels of THC, CBD or other cannabinoids. researchers have ever looked for them. One “Our main goal,” says Lumba, “is to make can- A more modern plant-breeding tech- that has been described comes from George nabis into a real horticultural crop.” Weiblen, a plant biologist at the University of Another decades-old practice for improving Minnesota in Saint Paul and one of the few aca- agricultural plants involves intentionally dou- demics in the United States who has a federal bling or tripling their genomes, which tends “THIS YEAR IS A licence to grow cannabis — but he’s restricted to give plants bigger cells, larger structural to just 50 plants at a time (see ‘The wide world features and greater yields of chemical com- TURNING POINT of weed’). It took him 12 years to determine the pounds. Domesticated wheat species, for exam- inheritance pattern of genes that affect drug ple, have 4–6 copies of their genome; sugar FOR CANNABIS content and to identify a genetic marker linked cane can have as many as 16. And although to the THC-to-CBD ratio5. That’s longer than most modern farmed plants had their DNA MOLECULAR it took Gregor Mendel to work out the laws multiplied simply through hundreds of years of heredity, Weiblen says. “Our programme of cultivation, there are ways to speed up the GENETICS.” is a poster child for the absurdity of cannabis process. All cannabis varieties characterized research in the United States.” so far have only two sets of the genome — all, A complete cannabis genome would make that is, except for a handful of plants growing at nique — one that dates back to the 1980s it easier to identify informative DNA mark- Canopy Growth Corporation in Smiths Falls. — is known as marker-assisted selection. It ers. But early efforts have yielded maps that are There, plant molecular geneticist Shelley involves finding genetic signatures associated patchy and incomplete, says Kevin McKernan, Hepworth at Carleton University in Ottawa and with a desirable trait — high essential-oil con- chief science officer and founder of Medicinal her former graduate student used a cell-cycle- tent, say, or automatic flowering under any Genomics in Woburn, Massachusetts, who disrupting herbicide to trigger five varieties of light condition. Scientists can then use DNA did some of the early work. He (see go.nature. cannabis to double their normal chromosome analyses to quickly ‘preview’ which seedlings com/2kpzgkc) and Page3 released maps

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A BUDDING TREND companies’ grow rooms move air over the plant Political movements to decriminalize cannabis for medical and recreational use have been gaining crowns. But in the thicket of leaves and branches in popularity. The result is a slowly shifting patchwork of regulations as many countries (and sometimes beneath, the air remains stagnant, leading to smaller jurisdictions) start to allow limited use and increasingly liberalize their stance. pockets of elevated temperature and humid- ity that breed fungal pathogens. The solution: a pressure gradient from floor to ceiling that ensures airflow along every surface of the plant. “It’s a very simple innovation,” Scanlon says. Deron Caplan, director of plant science at Flowr in Lake Country, completed a PhD this Fully legal year in which he systematically determined Medical optimal rates of fertilizer supply at various Illegal 9,10

stages of cannabis production and best ANALYTICS RESEARCH/BDS SOURCE: ARCVIEW MARKET Federally illegal, practices for propagating the plant clonally but allowed for 11 recreational or through cuttings . “It’s very crude, incremen- medicinal use tal advancements that we’re making,” says Mike on a regional Dixon, an agricultural scientist at the Univer- basis sity of Guelph and one of Caplan’s graduate advisers. But it’s slowly helping to phase out the homespun practices that persist throughout much of the industry, he says — “what I kindly refer to as anecdotal bullshit”. independently in 2011. “They’re train wrecks,” described a hormone recipe for inducing shoot Some of the holdovers of illegal cultivation says McKernan. But that’s changing. And thanks formation that, it says, works more than 80% of are on display at Beleave, a cannabis producer to an international trend toward less restrictive the time7. Yet, others say that they can’t get the in Hamilton, where master grower Shane laws around cannabis (see ‘A budding trend’), protocol to work on their own varieties. “There Whelan-Stubbs has persevered with some many weed companies are now investing in are tens of labs that are now working on making practices for 20 years, first in a bedroom cup- genetics research. the protocol more efficient,” says Leor Eshed- board, then in basements, warehouses and now Williams, a plant developmental geneticist at the a legitimate business. Whelan-Stubbs is open DANK DNA Hebrew University of Jerusalem. to the science, and Beleave will soon start col- “This year is a turning point for cannabis For many in the industry, however, any sug- laborating with a team at Guelph, where scien- molecular genetics,” says C. J. Schwartz, gestion of genetic modification is an anathema. tists hope to open the country’s first dedicated founder and chief executive of Sunrise Genet- Besides, says Ethan Russo, director of research academic centre for cannabis research some ics in Fort Collins, Colorado — one of at least and development at the International Canna- time next year. Still, Whelan-Stubbs continues six companies that say they have come up with bis and Cannabinoids Institute in Prague, “this to water his plants by hand. “The plants, at the fine-scaled genome maps. They have not yet plant is so malleable that a lot of these genetic end of the day, need love,” he says. been published yet in a peer-reviewed journal, modifications are really unnecessary”. Modern Page would argue that they also need science. but McKernan posted a preprint of his map on selective-breeding strategies, he argues, should But the burgeoning research that he’s been a 10 October6 and Schwartz expects to make his suffice — and those methods needn’t require part of likewise would have long ago withered sequence public by the end of the month. without cannabis at its centre. “We think of it in Then there are scientists who are hoping to many ways as a drug or a pharmaceutical,” he engineer new properties into the plant. At Can- says, “but can’t forget that it’s the plant that’s at opy Growth, research and development man- the heart of this revolution. It all comes down ager Katya Boudko worked with molecular “THE PLANTS, AT to a plant.” ■ biologist Douglas Johnson at the University of Ottawa to develop a gene-silencing technology THE END OF THE DAY, Elie Dolgin is a Canadian-born science that prevents expression of the THC-synthesis journalist in Somerville, Massachusetts. gene. Boudko expects plants to compensate by NEED LOVE.” 1. Günnewich, N. et al. Nat. Prod. Commun. 2, 223–232 boosting their levels of CBD — or, she says, “it (2007). could potentially produce other cannabinoids 2. Gagne, S. J. et al. Proc. Natl Acad. Sci. USA 109, that the world doesn’t even know about yet”. genetic markers. In collaboration with Mark 12811–12816 (2012). Boudko has yet to fully test this theory, Lewis, president of Napro Research in Westlake 3. van Bakel, H. et al. Genome Biol. 12, R102 (2011). 4. Sawler, J. et al. PLoS ONE 10, e0133292 (2015). however. That’s because she hasn’t managed Village, California, Russo has used chemical 5. Weiblen, G. D. et al. New Phytol. 208, 1241–1250 to grow fully fledged plants from genetically profiling to create dozens of cannabis varieties (2015). modified tissue — and nor have many others. with unique properties and elevated yields8. 6. McKernan, K. et al. OSF Preprints https://doi. org/10.31219/osf.io/7d968 (2018). Because seeds or clippings cannot be geneti- 7. Lata, H., Chandra, S., Khan, I. A. & Elsohly, M. A. cally modified in a consistent and predictable HASHING OUT THE BASICS Planta Med. 76, 1629–1633 (2010). way, scientists need to culture plant tissue and Elsewhere, researchers are looking to control 8. Lewis, M. A., Russo, E. B. & Smith, K. M. Planta Med. 84, 225–233 (2018). coax it into producing roots and shoots after and fine-tune environmental conditions at 9. Caplan, D., Dixon, M. & Zheng, Y. HortScience 52, the genes have been manipulated. Often, sci- various stages of the growth cycle. Such tweaks 1307–1312 (2017). entists can get the cellular masses to produce could prove important for maximizing profits 10. Caplan, D., Dixon, M. & Zheng, Y. HortScience 52, 1796–1803 (2017). fine root hairs, but the shoots have proved par- from costly indoor growing operations that are 11. Caplan, D., Stemeroff, J., Dixon, M. & Zheng, Y. Can. ticularly problematic. a major source of high-end cannabis in Canada. J. Plant Sci. 98, 1126–1132 (2018). In 2010, a team from the University of Missis- At CannTx Life Sciences in Puslinch, operations 12. Hudak, J., Ramsey, G. & Walsh, J. Uruguay’s Cannabis Law: Pioneering a New Paradigm (The sippi in Oxford, where for 50 years researchers head Jeff Scanlon and his colleagues developed Brookings Institution, 2018). have grown all the cannabis used for govern- a system for air circulation. 13. Welling, M. T., Liu, L., Shapter, T., Raymond, C. A. & ment-backed health studies in the United States, Scanlon showed that the fans found in most King, G. J. Euphytica 208, 463–447 (2016).