Tag Archives: genetic

Shimadzu & Front Range Biosciences Announce Partnership

By Cannabis Industry Journal Staff
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According to a press release, Shimadzu Scientific Instruments and Front Range Biosciences (FRB) announced a partnership where they will establish the FRB Hemp Science Center of Excellence. The center will focus on genetics, biobanking, breeding and analysis, all with Shimadzu instrumentation. The center will host scientists performing chemical and genetic analytical research to “support the development of new hemp varieties for the production of cannabinoids, terpenoids and other compounds for medical and wellness applications; lipid, wax and protein ingredients for food and cosmetics applications; and fiber for industrial applications.”

Dr. Jon Vaught headshot
Dr. Jon Vaught, CEO of Front Range Biosciences

Located at FRB’s new headquarters in Boulder, CO, the new center will allow for collaborative efforts between researchers from the public and private sectors like academic, nonprofit and government agencies. The center will expand FRB’s collaboration with the University of Colorado at Boulder. Researchers at other institutions can apply for grants to support students, postdoctoral candidates and other researchers at the new center.

Dr. Jonathan Vaught, CEO of FRB, says they’re honored to work with Shimadzu and their instrumentation. “Front Range Biosciences is honored to partner with the world-class team at Shimadzu. Combining their innovative and proven cannabis and hemp analytical instrumentation solutions with our next-generation breeding program, we will be well positioned to continue advancing the industry with data-driven science so we can harness the incredible potential of this versatile plant for therapeutic, wellness, nutrition and industrial applications,” says Dr. Vaught. “We are confident that with Shimadzu’s cutting-edge technology, we will be able to drive forward-thinking solutions in this growing industry to better serve farmers, producers and consumers.”

New Cannabis Science Course Offered at University of Colorado at Boulder

By Cannabis Industry Journal Staff
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According to a press release, the University of Colorado at Boulder is offering a new course focused on cannabis science through the Continuing Education program at the university during the upcoming Summer and Fall semesters.

The class is called Modern Cannabis Science and will involve a lot of genetic research. The course is sponsored by the Agricultural Genomics Foundation, a non-profit dedicated to scientific research and education in cannabis. In the press release, they describe the course as meant for students who are well informed, but “seek a deeper appreciation of scientific advancements in cannabis genetics.”

Here’s a snapshot of what students can expect to learn from it:

In Modern Cannabis Science, we will explore the range of Cannabis research currently available covering topics such as evolutionary history and global distribution, sex chromosomes, genetic contribution to chemotype, and analyses to aid law enforcement and forensic investigations. We will examine how genetic data allow us to understand relationships between strains and common categories in the Cannabis genus, and why this is important for breeding, policy-making, and medical purposes.

The press release suggests students who enroll can expect to use this knowledge in the cannabis industry. “For example, a budtender will be able to more accurately recommend strains to users,” reads the press release. “Similarly, medical personnel will more fully understand the relationship between strains, the compounds they produce, and how to properly advise Cannabis patients.”

For more information, take a look at the course here.

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Confront Poor Medicinal Cannabis Policies to Save Lives

By Dr. Jordan Zager
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For me, the opioid epidemic was never a theoretical crisis. The mounting lives lost to overdoses weren’t just numbers in news reports to me, but names. A high school lab partner, little league teammates, a cook at my first restaurant job and others in my hometown were lost to the epidemic. By the time I graduated high school, seven people in my life died due to complications arising from opioid use.

What’s not lost on me now, after earning my PhD in plant biochemistry and founding a startup focused on bringing consistency and scientific credibility to the cannabis industry, is how a stigma around medicinal cannabis seems like such a contributing factor in their deaths.

Cannabis, although fully illegal in only eight U.S. states, still qualifies as a Schedule 1 drug on the federal level, legally equivalent to LSD or heroin. Crystal methamphetamine and cocaine as Schedule 2 drugs have lower penalties and even have federally approved medical applications. This is where we’ve failed as a broader scientific community.

The reason is this: Medicinal cannabis produced from the same genetic replicates, but grown in separate locations, or even different seasons, will possess different bioactive compounds. In short, their effect on patients will be different depending on the various bioactive compounds produced by the plant. Prescription medications do not come with that major caveat.

Dr. Jordan Zager, CEO and co-founder of Dewey Scientific

There’s a quality assurance problem, compounded by a lack of science that’s been shackled by the criminalization of cannabis since 1937.

We do know that the primary benefits of cannabis are three-fold: First, there’s pain management, as 28 well conducted randomized clinical trials (RCTs) have documented that cannabinoid agents are effective analgesics for chronic pain. Second, while potentially psychologically addictive, so people may desire the “high” produced by cannabis, THC is not chemically addictive and does not create a biological desire for the drug, much like the craving induced by the absence of, say, cocaine or heroin has on regular users. And finally, patients cannot overdose.

As a scientific community, there are three things we need to start doing today to change the narrative around medicinal cannabis and help bring this safer alternative therapy to more people. We need to provide a larger body of evidence about the benefits. We need to drive increased consistency in cannabis products themselves. And we need to confront stigmas rooted in misinformation. The sooner we can succeed here, the sooner we can hope for a day when we see fewer devastating opioid overdoses and deaths.

I am driven by a vision for a future when people can have access to safe, trusted and consistent cannabis for their medical and recreational uses, and we as a society are able to fully realize the therapeutic benefits of this amazing plant. As scientists, my colleagues and I are committed to doing our part to bring the credibility and advancements that will help this vision become a reality.

Using tools rooted in science–including functional genomics and secondary metabolite pathway expression profiling–cultivators can learn to fully “know” the plants they grow and hone in on producing the same bioactive compounds and in the same ratios that show medicinal promise. Cultivators can learn the genetic effect that their facility has on their genetics and why those genetics lead to a different chemical profile when grown elsewhere. Together, we can identify the driving factors of what makes a variety help with whatever ailment you are trying to treat.

I’m buoyed by data that shows states that have legalized and provided access to recreational cannabis have between 20% and 35% fewer reported opioid deaths, and lower rates of opioid prescriptions. But more needs to be done. I plan to become a more vocal voice, advocate for sound science, consistency in medical cannabis and better access to natural plant-based medicines without the stigma of yesteryear.

The time has come for our policy makers to step up. We cannot afford to just be observers when the cost of remaining on the sideline is measured in lives.

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Clean Grow Still Failing? Check for Endophytic Mold

By Bernie Lorenz, PhD
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The journal Frontiers in Plant Science recently shared an important article from researchers at Simon Fraser University in British Columbia, highlighting the “Pathogens and Molds Affecting Production and Quality of Cannabis Sativa.”

As a chemist focused on the science of preventing and mitigating mold in greenhouse and indoor cannabis grow facilities, this piece was fascinating to me. Like many others, it details and explains prevalent mold like Penicillium, Cladosporium and Aspergillus – things I see in grows every day.

But wait, there’s more fungi

The research and resulting article also brought up another type of fungi – endophytic mold. Endophytic mold usually lives symbiotically with plants, or is at least beneficial for both plant and fungi.

But not always.

In the past, the industry has believed that damaging mold spores were found on the outside of the flower. When moved, that flower would release the spores and send them flying – often creating massive cross-contamination issues for indoor grows.

Hope Jones, PhD, CEO of Adivina & ECS

“While cannabis is an incredibly powerful plant in terms of its medicinal properties, it is unfortunately highly susceptible to many pest and pathogens,” says Hope Jones, PhD, CEO, Adivina & ECS. “And it is this susceptibility that is so challenging to many inexperienced or undisciplined grow operations.”

Now, however, we know that there’s another culprit to add to the list: the inner parts of the plant can also be a source of endophytic cross contamination and mold.

Since it grows inside of the plant, this fungus creates high spore counts that can cross contaminate from outside, into the flower.

Treating mold in a facility

Here’s the good news:

This seemingly bad news – that there’s a new fungus to worry about, and it is inside the flower – may actually help cannabis grows struggling with mold, and those who are following the proper protocols already.

A petri dish of mold growth from tested cannabis Photo credit: Steep Hill

Effective mitigation protocols can include things like treating HVAC systems, controlling humidity, using products like chlorine dioxide to treat irrigation lines, enforcing protective clothing and shoe covers for employees, reducing the amount of in-and-out for employees around grow rooms.

These are important upstream and environmentally-focused integrated pest management (IPM) programs that will usually keep facilities clean and relatively mold-free.

But if these programs are in place, and there’s still an issue, Endophytic fungi may be to blame.

If you are having ongoing mold issues but have ruled out cross-contamination and a facility without proper protocol, look to the mother plant.

“Small mistakes in agricultural practices are amplified with cannabis,” Dr. Jones continues. “And today’s propagation practices of traditional cloning add to this vulnerability. Cannabis is an annual plant and by keeping mothers in a perpetual state of vegetative growth for years, and taking repetitive cuttings produces clones in a highly stressed state. This stressed state diminishes genetic potential and weakens a plant’s ability to fight disease and pests.”

Testing for and addressing endophytic fungi

If these concerns are ringing a bell, remember, there is also a way to test for Endophytic mold.

Checking cuttings from suspected mother plants over a period of time is the best way to see if the Endophytic mold is present.

A section of the mother plant cutting is placed into a solution (for example, as outlined by the article, a very concentrated hypochlorite followed by 70% Ethanol) that will kill all of the microorganisms that are present on the surface of the plant tissues.

A large tissue culture facility run in the Sacramento area that produces millions of nut and fruit trees clones a year.

From there, an unadulterated dissection of the internal tissues can be extracted and cultured for quantification and identification of endophytic fungi.

“Tissue culture offers a form of genetic rebooting returning the plant to its natural genetic potential and thereby strengthening its natural ability to defend against environment assault,” says Dr. Jones. “It also allows the breeder to conduct pathogenic disease testing which provides the entire industry with a higher level of scientific certainty and analysis.”

If you find this mold inside of the mother plant, your facility’s mold problem could be a systemic issue, not an environmental one.

If you do find that Endophytic mold is causing issues, of course, you may have to destroy the mother plant.

This should not mean the end of a strain. Tissue culture on a cutting is an option that can eliminate the unwanted fungi and save the genetics. Using those genetics to regrow a mother will start fresh and avoid the intrinsic mold that was plaguing the strain prior.

Growing knowledge

The practice of checking mother plants for Endophytic mold is not yet commonplace in cannabis, but the hemp business is leading the way.

They’re testing to create very clean plants, so you don’t have issues during cultivation.

Major growers in the U.S. could save millions in lost harvests with mold mitigation. If your current IPM program isn’t doing the trick, you may want to follow in hemp’s footsteps and look inside the plant.

Large Scale Cultivation Planning: 4 Important Factors to Consider

By David Perkins
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Before you begin any large-scale cultivation project, you must necessarily consider the four factors highlighted below, among many others, to ensure your cultivation is successful. Failure to do so will cost you greatly in both time and money, and ultimately could lead to failure. While the four areas highlighted below may be the most important considerations to address, you should hire a cultivation advisor to determine the numerous other considerations you must deal with before you begin.

1. Genetics

Genetics will play a huge role in your cultivation plan, as they can ultimately make or break the success of your business. Access to quality, verified genetics will greatly affect your profits. All cannabis genetics grow differently and may require different conditions and nutrients. Further, consumers in today’s regulated market have greater awareness; they are much more knowledgeable about genetics and able to discern between quality cannabis versus commercially produced cannabis.

Market trends will dictate whether or not you’ll ultimately be able to sell your harvest at market rate. You need to project out at least one year in advance the genetics you will be growing. But often it is impossible to predict what consumers will be purchasing a year in advance so this part of your cultivation plan should be well thought out. Further compounding this difficulty is the fact that it may take six months to ramp up production of any given variety.

Genetics that are popular now may still be popular next year, but that also means there will be more competition for shelf space, as more competitors will also likely be growing these same genetics. Therefore, don’t rely on only one trendy variety as the bulk of your selection for the year, no matter how popular it is at the moment. Producing a single variety as the bulk of your crop is always risky, unless you have a contract with a sales outlet, in advance, for a set quantity of that one particular variety. Diversity in your genetics is beneficial, when chosen correctly.

Making proprietary genetics from your own seed collection can give you a big advantage in today’s competitive market. Having a variety with a distinct, unique and desirable smell, taste, effect or cannabinoid profile will allow you to distinguish your brand amongst others. Entire brands have been built off of a single variety: Cookies and Lemontree are two examples of companies that have done this. All it takes is one really good variety to attract a lot of attention to your brand. Having your own breeding project on site will allow you to look for and identify varieties that work for you and your business model, and ultimately will help to distinguish your brand apart from others.

Only buy seeds from reputable breeders! Any new varieties that you are going to be cultivating should be tested out at least three times, on a small scale, before being moved into a full production model. If you are growing from seed there is always the potential for your crop to get pollinated by male plants or hermaphrodites that went unnoticed, and therefore, they could be a potential risk to your entire harvest. Treat them accordingly, i.e. by cultivating them on a small scale in a separate, enclosed area.

Buying clones from a commercial nursery can be risky. Genetics are passed from one grower to another haphazardly, and names are changed far too easily. This can create a lot of confusion as to what variety you are actually purchasing and whether you are getting the best version of the genetics. Just because a clone is called “sour diesel” doesn’t mean you’re actually getting the real, authentic sour diesel. And to further complicate things, the same clone grown in different environments can produce a noticeable difference in flavor, smell and effect depending on your cultivation method. Always try your best to verify the authenticity of the genetics you purchase. Ask about the history and origin of the particular genetics you are purchasing. Better yet, ask for pictures, physical samples, and most importantly, certificates of analysis from a laboratory, indicating the potency. In many states anything under 20% THC is going to be hard to sell, while anything over 30% will easily sell and command the highest price. It’s a good idea to have a laboratory test the terpene profile in order to verify a variety is actually what the seller purports it to be.

Knowing the source of your genetics is imperative. It will help ensure that you actually have the variety that you were intending to grow, and therefore, allow you to achieve your intended results. Knowing what varieties you are going to cultivate, before you grow them, will also give you a better idea of the ideal growing conditions for that specific variety, as well as what nutrients will be required to achieve optimum output.

2. Automated Watering Systems

Installing an automated watering system, during build out, will by far be the most cost-effective use of your money, and will save you the most amount of time in labor. An automated watering system, commonly referred to as a “drip system” or “drip irrigation,” is necessary regardless of whether you are cultivating indoors or outdoors; it will allow you to water multiple different areas at once, or only water a few specific areas of the garden at one time. Hand watering a 22,000 square-foot cultivation site will take one person eight hours every single day, on average, to maintain. However, a properly designed drip system can water an entire large-scale garden in a couple of hours, without any employees, record all the relevant data and notify you if there is a problem. This enables you more time to spend closely inspecting the plants to ensure there are no bugs or other problems present, and that your plants are healthy and thriving. This attention to detail is necessary if you want to have consistent success.

Larger scale cultivation requires bigger and more expensive equipment.

Automated watering systems not only save a great deal of time but also eliminate the possibility of human error, like over watering, which can kill an entire crop quickly. There aresoil moisture sensors  that can be placed in the soil to regulate the supply of water to the plants in a precise manner. Without an extremely skilled, experienced work force, damage to plants due to over watering is very common. A drip system will reduce the threat of human error by ensuring delivery of precisely the correct amount of water and nutrients to each plant every single time they are watered.

Not all drip systems are created equally. There are different types of automated watering systems. Designing the right drip system for your cultivation site(s) can be complicated. Make sure you do your research, or better yet, work with a cultivation advisor who has experience with automated irrigation systems in conjunction with a licensed plumber, to ensure you are installing the best system for your particular set up.

Adding a fertilizer injector to your drip system can further increase the efficiency of your operation and save you money on nutrients by using only what you need and ensuring correct application. Again, automating this process will save you time and money, and reduce the threat of human error.

3. Nutrients

The types of nutrients you use and the amount of nutrients you use, are going to directly affect the quality of your cannabis flower. Conventional agriculture and Dutch hydroponic cannabis cultivation have always used salt-based fertilizers. However, they can be toxic for the plant in high amounts. While cheap and easy to use, salt- based nutrients are made in big factories using chemical processes to manufacture. They are not good for the environment, and overall, they produce an inferior product. The highest quality cannabis, is grown with organic living soil. Although seemingly contrary to popular knowledge, when done properly, cultivating in organic living soil is more cost effective than using powdered or liquid salt-based fertilizers.

Yield and quality depend on the skills of the cultivator, more than the method they are using. Having healthy plants from the start, will always yield better results, no matter what way they were grown. In my 20 years of experience I have seen plants grown in balanced living soil yield just as much as plants grown with synthetic nutrients. Further, the quality is not comparable.

Controlling your clone supply can ensure they are healthy

Always remember, it is the quality of your flower that will determine the price it is sold for, not the yield. Even if you produce more overall weight of chemically grown cannabis, if nobody wants to purchase that product, then you are going to yield far less profit than another company growing in the same amount of space using organic practices that yield a higher quality product.

The difference in quality between plants grown in balanced living soil versus any other method of cultivation is undeniable. It is really easy to post a pretty picture of a flower on Instagram but that picture doesn’t tell you anything about what went into producing it. When flower is produced using chemical nutrients, it is likely going to be harsh and not enjoyable to smoke. Lesson learned: don’t judge a bud by an Instagram photo! There is a stark difference between cannabis grown using synthetic nutrients versus cannabis grown in living soil. Once you’ve experienced the difference you will never want to consume cannabis that is grown any other way.

4. Plant Propagation

Having the ability to propagate your own clones, from mother plants that you have cultivated, can save you a staggering amount of money. In some states, having a cultivation license allows you to produce your own clones for your cultivation, while having a nursery permit will allow you to sell clones for commercial sales to other companies. The average price of a wholesale clone is around eight dollars. If you require 5000 plants for every harvest, that’s a $40,000 expense you must bear, every grow cycle. This can obviously add up quickly. And as previously mentioned there’s the risk of purchasing inferior genetics or unhealthy plants, both of which greatly affect your profit margins.

On the other hand, the cost of materials and labor to produce a healthy clone can be as low as one dollar when using advanced cloning techniques. Controlling your clone supply can ensure they are healthy and allow you to know exactly what you are growing each time. Further, it doesn’t take a lot of space to propagate your own cuttings. In a 400 square-foot space one could produce between 5,000 to 10,000 clones per month, all of which could be maintained by one person depending on your situation.

And last but definitely not least, the most important thing you can do to ensure the success of your cultivation, is hire an experienced knowledgeable grower who is passionate about cannabis. The success of your company depends on it. You need someone with the knowledge, experience, and skills to make your cultivation dreams a reality. You need someone who can plan your build-out and cultivation to ensure success from the start. And you need someone with the skills to handle the multitude of inevitable problems that will arise in a cost effective and efficient way.

These are just some of the many considerations you must account for when planning a large scale grow in the regulated market. An experienced cultivation advisor can help you with these, and many other considerations you will need to contend with before you begin your grow. Creating a well thought out plan at the outset can end up saving you thousands, if not hundreds of thousands of dollars down the road.

Khyrrah-Cymone Shepard
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Challenges in Cannabis Genome Sequencing for Genetic Tracking and Traceability

By Khyrrah-Cymone Shepard
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Khyrrah-Cymone Shepard

Genome sequencing has made remarkable strides since the initiation of “The Human Genome Project” in 1990. Still, there are many challenges that must be overcome before this methodology can reach its fullest potential and be useful in serving as a method of Cannabis sativa genetics verification and tracking throughout the cannabis supply chain. Several major milestones that must be realized include end-to-end haploid type (single, unpaired set of chromosomes instead of complete paired set or “diploid”), long read, resolved genome sequences at a reasonable cost within a reasonable timeframe and with confidence in accuracy (Mostovoy et al.). These genomes are typically generated as shorter reads that are then scaffolded (Fig 1.) or matched to reference genomes in order to build a longer continuous read. While shorter sequencing reads indeed lower the cost barrier for producing more genomic data, it has created another issue as a result of this short-read technology.

Figure 1: Four sets of sequencing data (long-read WGS, Hi-C, optical mapping, and short-read WGS) were produced to generate the goat reference genome. A tiered scaffolding approach using optical mapping data followed by Hi-C proximity-guided assembly produced the highest-quality genome assembly. (Bickhart et al.)

There are two main issues with the more affordable short read sequencing methodology, the first being that sequential variants are typically not detected, especially if they involve a ton of repeats/inverted repeats, due to the limitation of the current referenced Cannabis genomes and the mapping process of the short-read sequences. This is especially unfortunate because larger variants can have up to a 13% variance within a diploid multichromosomal genome, such as Cannabis sativa, and this variance is thought to largely contribute to disease in various species, or maybe terpene profile in Cannabis sativa. Not being able to detect these variances with more affordable sequencing methodologies is particularly problematic and reference genomes produced with short read sequences are typically highly fragmented. The second limitation is the inherent errors, gaps and other ambiguities associated with taking tons of short read sequences and combining them all, like a jigsaw puzzle, in order to draft the larger genomic picture. While there is software with algorithms to assist in deciphering raw sequences, there is still much more work to be done on this challenge, considering that cannabis genome sequencing is new genomics territory. Unfortunately, as researchers seek higher and higher levels of data quality, shortcomings of this type of sequencing technology begin to become apparent. This sort of sequencing methodology relies heavily on reference sequences. This isn’t much of an issue with microbial genomes, which tend to be rather short and typically have one chromosome, however, when seeking to analyze much longer genomes with multiple diploid chromosomes and tons of mono and dinucleotide repeats, problems arise (English et al.).

Figure 2: Blockchain Digital Stamping Certificate which publicly documents the date and time of the completion of this work. (Mckernan – Crypto Funded Public Genomics)

The other category of sequencing is long read sequencing. Long read sequencing is as it sounds, the deciphering of much longer DNA strands. Of course, the technology is limited by the quality of the DNA captured, therefore, special high molecular weight DNA extraction protocols must be deployed in order to obtain the proper DNA quality (Fig. 3). Once this initial limitation is overcome there is the stark cost of long read sequencing technology. PacBio without a doubt makes one of the highest quality long read sequence generating instruments that has ever graced the field of biotechnology, but due to the steep price tag of the machine, progress in this field has been stifled simply because it just isn’t affordable and the read depth for mammalian and plant genomes is currently almost completely prohibitive until read lengths double in length for this instrumentation. In order to produce what is considered to be a “validated genome” both short read and long read sequencing methodologies are combined. Long read sequencing data is used to produce the reference contigs because they are much easier to assemble, then short read sequencing is scaffolded against the reference contigs as a sort of “consensus validation” of the long read contigs.

Figure 3: Depiction of various DNA high molecular weight DNA quality captured during cannabis genome submission project. (Mckernan – Crypto Funded Public Genomics)

Despite the shortcoming of utilizing short read sequencing technology for analysis of the cannabis genome, it is still useful especially when combined with other longer read sequencing technologies or optical mapping technologies. Kevin McKernan, chief scientific officer of Medicinal Genomics, has been working feverishly to bridge the information gap between the cannabis genome and other widely studied plant genomes. As a scientist that worked on the Human Genome Project in 2001, McKernan has a demonstrated history of brilliance in the field of genomics. This paved the way for him to coordinate the first crypto funded and blockchain notarized sequencing project (DASH DAO funded) (Fig. 2), which was completed in 60 days, and surprisingly showed that the cannabis genome is over 1 billion bases long which is 30% larger than any cannabis genome submitted prior to his work. By reaching the standard of 500kb N50 set forth by the Human Genome Project, Kevin McKernan was able to see new aspects of the cannabis genome that were not visible due to the fragmented genomic data previously generated. Information such as a possible linkage of THCA synthase and CBDA synthase genes is crucial when seeking to use the cannabis genome for verification and tracking purposes. This is because special linkages can be considered a type of “genetic marker” that may be used to differentiate cannabis cultivars and lineages. There are many types of genetic markers, including SNP (single nucleotide polymorphisms), VNTR (variable number tandem repeats) and even patterns of gene expression. Funding and recording of cannabis genomics must be further developed in order for potential markers to be identified and validated via larger scale genome-wide association studies.

These technologies, when combined, often reduce the number of scaffolds while increasing the percent of resolved genome by filling in gaps within the drafted genome. Nanopore sequencing is an especially interesting and innovative sequencing technology that is useful in many ways. One of the most powerful uses of this technology is its ability to upgrade the quality of draft and pushed genomes by resolving poorly organized genomes and genomic structure for a fraction of the time and cost of other long read sequencing platforms (Jian et al.), making it an excellent candidate for solving cost and time constraints. Nanopore’s portability and convenience makes it a real-time solution to solving genetics-based problems and questions. A notable use of this technology is recorded during an epidemiological outbreak in Africa, its proof of concept in pathogen detection in space, and its ability to detect base modifications during sequencing process. Even still there are more uses to this exciting technology and it has the potential to elevate cannabis genomics and the field of genomics entirely, while remaining portable and expeditious. A shortcoming of the Nanopore sequencing platform is its low sequencing coverage, which makes this platform inefficient for applications like haplotype phasing and single nucleotide variant detection due to the number of variants to be detected being smaller than the published variant-detection error rates of algorithms using MinION data. Single nucleotide variants can be considered to be genetic markers, especially markers for disease, so this is what inhibits Nanopore from resolving our cannabis genome sequencing problems, as of today.

There are genetic markers to discover, molecular biology protocols to optimize, and industry wide potential for exciting collaborationMany algorithmic problems seem to occur due to input data quality. Typical input data quality suffers as the reads get longer and the sequencing depth gets shorter, resulting in not enough data being generated by the sequencing to provide confidence in the genome assembly. To mitigate this, scientists may decide to fractionate a genome, sequence it, or they may clone a difficult to sequence region with highly repetitive regions in order to produce reads with greater depth and thus resolve the region. They can then perform single molecule sequencing to resolve genome structure then determine and confirm the place of the cloned region. Thus, it seems that the best solution to the limitation of algorithms is to be aware of sequencing platform limitations and compensate for these limitations by using more than one sequencing platform to obtain enough pertinent data to confidently produce authentic, “validated” genome assemblies (Huddleston et al.). With input data being critical in producing accurate sequencing data, standardization of DNA isolation protocols, extraction reagents and any enzymes utilized may be deemed necessary.

To conclude, the field of cannabis genomics is teeming with opportunities. There are genetic markers to discover, molecular biology protocols to optimize, and industry wide potential for exciting collaboration. More states will need to take into account the lack of federal government research grant availability and begin to think of creative ways to get cannabis science funds to continue the development of this industry. Specifically speaking, developing a feasible method for genetic tracking of cannabis plants will require improvements within the availability of sequencing technology, improvements in deploying the resources to these projects in order for them to be completed expeditiously, and standardization/validation of methods and SOPs used in order to increase confidence in the accuracy of the data generated.

A special thank you to all of my cannabis industry mentors that have molded and elevated my understanding of current needs and applied technologies within the cannabis industry, without you there would be no career within this industry for me. You are immensely appreciated.


Citations

Bickhart, D. M., Rosen, B. D., Koren, S., Sayre, B. L., Hastie, A. R., Chan, S., . . . Smith, T. P. (2017). Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome. Nature Genetics,49(4), 643-650. doi:10.1038/ng.3802

English, A. C., Salerno, W. J., Hampton, O. A., Gonzaga-Jauregui, C., Ambreth, S., Ritter, D. I., . . . Gibbs, R. A. (2015). Assessing structural variation in a personal genome—towards a human reference diploid genome. BMC Genomics,16(1). doi:10.1186/s12864-015-1479-3

Huddleston, J., Ranade, S., Malig, M., Antonacci, F., Chaisson, M., Hon, L., . . . Eichler, E. E. (2014). Reconstructing complex regions of genomes using long-read sequencing technology. Genome Research,24(4), 688-696. doi:10.1101/gr.168450.113

Jain, M., Olsen, H. E., Paten, B., & Akeson, M. (2016). The Oxford Nanopore MinION: Delivery of nanopore sequencing to the genomics community. Genome Biology,17(1). doi:10.1186/s13059-016-1103-0

Mostovoy, Y., Levy-Sakin, M., Lam, J., Lam, E. T., Hastie, A. R., Marks, P., . . . Kwok, P. (2016). A hybrid approach for de novo human genome sequence assembly and phasing. Nature Methods,13(7), 587-590. doi:10.1038/nmeth.3865

The First Map of the Cannabis Genome

By Aaron G. Biros
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Sunrise Genetics, Inc., the parent company for Hempgene and Marigene, announced last week they have successfully mapped the cannabis genome. The genome map was presented at the 26th Annual Plant and Animal Genome Conference in San Diego, CA during the panel “Cannabis Genomics: Advances and Applications.”

According to CJ Schwartz, chief executive officer of Sunrise Genetics, the full genome map will allow breeders to develop strains using DNA sequence information to complement phenotyping. “In this way a breeding program can be guided by the breeder versus blindly as it is for just pheno-hunting,” says Schwartz. “At the DNA level, we can identify what version of a set of genes a plant contains, and make predictions as to the phenotype, without ever growing the plant. As we make more and more gene markers, we have more genes to track, and breeding becomes more rapid, efficient and precise.” Schwartz says this is essential for breeding stable, repeatable plants. “A commercial strain will be grown in different environments, with solid genetics, the phenotype will mostly stay true, a term we call Genetic Penetrance.”

Ancestry-painted chromosomes for marijuana Image: Chris Grassa / Sunrise Genetics

Determining a plant’s DNA can be extremely valuable and completing the map of the genome now makes this more precise. It can serve as a point of proof, according to Schwartz, providing evidence of lineage in a breeding project and confirming the uniqueness and identity of a strain. The genome map can also allow breeders to select specific genes to develop custom strains. And in addition to all that, it provides legal protection. “Knowing your plants DNA code is the first step to being able take action so no one else can protect it,” says Schwartz. “Well documented evidence in the development of a customized strains is essential to maintaining control of your plant and keeping those you distrust (big pharma) away, many of which have minimal interest in the whole plant anyhow.”

CJ Schwartz, chief executive officer of Sunrise Genetics

Schwartz says this project took them roughly 18 months to wrap up. “One of the biggest problems was just finding the right plants to grow,” says Schwartz. “In addition we used some emerging technologies and those had some challenges of their own.” According to Schwartz, a key aspect in all this was finding the right collaborators. They ended up working with CBDRx and the plant biology department at the University of Minnesota, where a DEA-licensed lab has been researching cannabis since 2002. “George Weiblen’s group at UM has been working on Cannabis for over a decade,” says Schwartz. “During that time they did repeated selfing to make highly inbred marijuana and hemp lines. The lines were instrumental in deterring the physical order of the genes.”

Ancestry-painted chromosomes for hemp Image: Chris Grassa / Sunrise Genetics

After finishing up some experiments, they expect to get the genome map published on public domain in less than a year, opening up their research to the general public and allowing breeders and growers to use their data. “This will be a very significant publication,” says Schwartz. “The genome assembly allows for the assimilation of all the currently incompatible Cannabis genome sequence datasets from academia and private companies,” says Schwartz. “Joining datasets from 1000s of strains, and from every continent, will generate an essential public resource for cannabis researchers and aficionados alike.” With a tool like this, we can discover the genes that help produce desirable traits. “This project is a major accomplishment for cannabis, bringing it on par with other important crops, providing a scientific tool to unravel the secrets of this incredibly versatile plant,” says Schwartz.

Sunrise Genetics is assisting cannabis businesses in evaluating strains and developing breeding programs, working with a number of customers currently to develop strains for many different specific traits. “We have the expertise to help select parental strains and guide the selection process at each generation using genotype and phenotype information,” says Schwartz. “Essentially we are bringing all the tools any modern plant breeder would use for improving strawberries to cannabis.”

Dr. Zacariah Hildenbrand
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Cannabis and the Environment: Navigating the Interplay Between Genetics and Transcriptomics

By Dr. Zacariah Hildenbrand
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Dr. Zacariah Hildenbrand

It is that time of year where the holidays afford us an opportunity for rest, recuperation and introspection. Becoming a new father to a healthy baby girl and having the privilege to make a living as a scientist, fills me with an immeasurable sense of appreciation and indebtedness. I’ve also been extremely fortunate this year to spend significant time with world-renowned cannabis experts, such as Christian West, Adam Jacques and Elton Prince, whom have shared with me a tremendous wealth of their knowledge about cannabis cultivation and the development of unique cannabis genetics. Neither of these gentlemen have formal scientific training in plant genetics; however, through decades of experimentation, observation and implementation, they’ve very elegantly used alchemy and the principles of Mendelian genetics to push the boundaries of cannabis genetics, ultimately modulating the expression of specific cannabinoids and terpenes. Hearing of their successes (and failures) has triggered significant wonderment and curiosity with respect to what can be done beyond the genetic level to keep pushing the equilibrium in this new frontier of medicine.

Lighting conditions can greatly impact the expression of terpenes (and cannabinoids) in cannabis.Of course genetics are the foundation for the production of premium cannabis. Without the proper genetic code, one cannot expect the cannabis plant to express the target constituents of interest. However, what happens when you have an elite genetic code, the holy grail of cannabis nucleotides if you will, and yet your plant does not produce the therapeutic compounds that you want and/or that are reflective of that elite genetic code? This ‘loss in translation’ can be explained by transcriptomics, and more specifically, epigenetics. In order for the genetic code (DNA) to be expressed as a gene product (RNA), it must be transcribed, a process that is modulated by epigenetic processes like DNA methylation and histone modification. In other words, the methylation of the genetic code can dictate whether or not a particular segment of DNA is transcribed into RNA, and ultimately expressed in the plant. To put this into context, if the DNA code for the enzyme THCA synthase is epigenetically silenced, then no THCA synthase is produced, your cannabis cannot convert CBGA into THCA, and now you have hemp that is devoid of THC.So what is the best lighting technology to enhance the expression of terpenes? 

With all of that being said, how do we ensure that our plants thrive under favorable epigenetic conditions? The answer is the environment; and the expression of terpenes is an ideal indicator of favorable environmental conditions. While amazing anti-inflammatories, anti-oxidants and metabolic regulators for humans, terpenes are also extremely powerful anti-microbial agents that act as a robust a line of defense for the plant against bacteria and pests. So, if the threat of microbes can induce the expression of terpenes, then what about other environmental factors? I am of the opinion that the combination of increased exposure to bacteria and natural sunlight enhances the expression of terpenes in outdoor-grown cannabis compared to indoor-grown cannabis. This is strictly my opinion based off of my own qualitative observations, but the point being is that lighting conditions can greatly impact the expression of terpenes (and cannabinoids) in cannabis.

A plant in flowering under an LED fixture

So what is the best lighting technology to enhance the expression of terpenes? Do I use full spectrum lighting or specific frequencies? The answer to these questions is that we don’t fully know at this point. Thanks to the McCree curve we have a fundamental understanding of the various frequencies within the visible light spectrum (400-700nm) that are beneficial to plants, also known as Photosynthetically Active Radiation (PAR). However, little-to-no research has been conducted to determine the impacts that the rest of the electromagnetic spectrum (also categorized as ‘light’) may have on plants. As such, we do not know with 100% certainty what frequencies should be applied, and at what times in the growth cycle, to completely optimize terpene concentrations. This is not to disparage the lighting professionals out there that have significant expertise in this field; however, I’m calling for the execution of peer-reviewed experiments that would transcend the boundaries of company white papers and anecdotal claims. In my opinion, this lack of environmental data provides a real opportunity for the cannabis industry to initiate the required collaborations between cannabis geneticists, technology companies and environmental scientists. This is one field of research that I wish to pursue with tenacity and I also welcome other interested parties to join me in this data quest. Together we can better understand the environmental factors, such as lighting, that are acting as the molecular light switches at the interface of genetics and transcriptomics in cannabis.

Applications for Tissue Culture in Cannabis Growing: Part 2

By Aaron G. Biros
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In the first part of this series, we introduced Dr. Hope Jones, who took her experience in tissue culture from NASA and brought it to the cannabis industry and C4 Laboratories. We discussed some of the essential concepts behind tissue culture and defined a few basic terms like micropropagation, totipotency, explants and cloning. Now let’s get into some of the issues with cloning from mother plants and the advantages that come with using tissue culture for propagating and cultivating cannabis.

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Dr. Hope Jones, chief scientific officer at C4 Labs

Taking cuttings from mother plants is arguably the most popular method of propagating cannabis plants. It is a process that requires significant real estate, resources and labor. “Moms can take up a great deal of space that is not contributing directly to production,” says Dr. Jones. “I know from experience that scaling up production and/or adding new strains to the production line requires significant time and resources to raise and maintain new healthy and productive mother plants.” Each mother plant produces a limited number of clones per harvest period and over the course of her life cycle.

By using tissue culture, a cultivator can generate an almost infinite number of clones from one plant cutting. With so many growers calculating their costs-per-square-foot, micropropagation is an effective tool to save space, labor and time, thus increasing profit margins. “Just to put it in perspective: Holly Scoggins’ book Plants From Test Tubes, cites a Day Lily cultivator who uses micropropagation to produce 1,000 plants in 30 square feet of shelf space each week,” says Dr. Jones. “Using conventional methods, one would need a half-acre to produce the same amount of plants.” Cultivators can produce a much greater number of plants-per-square-foot by using micropropagation effectively.

Damage from whiteflies, thrips and powdery mildew is all visible on this sick plant.

Early Health & Vigor

Most tissue culture methods use sterilized vessels that contain sugar-rich media to support growth of plantlets before they can photosynthesize on their own. “The media is prepped, poured into vessels, and placed in an autoclave (or pressure cooker) where it is subjected to high temps and pressure to achieve proper sterility.”

The sterile environment and rich growth media supplies plantlets with an abundance of everything they need. “When plantlets emerge from culture, they are pathogen-free, with a stockpile of food and nutrient reserves that support rapid growth and vigor, superior to conventional cuttings,” says Dr. Jones.

Stress & Disease

As any grower knows, mother plants can sometimes experience stress and disease. This might come in the form under or over-watering, heat stress, spider mites, whiteflies, mold and viruses. “Any stress or infection that a mother plant is subjected too can impact progeny health and productivity in a couple of ways,” says Dr. Jones.

Powdery mildew starts with white/grey spots seen on the upper leaves surface
Tobacco Mosaic Virus symptoms can include tip curling, blotching of leaf mosaic patterning, and stunting.

For example, diseases like powdery mildew and tobacco mosaic virus are often systemic, meaning that pathogens have spread to almost every tissue in the plant. Once infected, it is impossible to completely eliminate pathogens from tissues. Therefore any cuttings made from a diseased mother plant, even if they look perfectly healthy, will also be infected and can eventually present disease symptoms like reduced productivity and/or plant death, according to Dr. Jones.

How does tissue culture get around this problem? Remember that explants (small tissue samples used as starting material) can be extracted from any part of the plant. Meristematic cells in shoot tips and leaves are the source of new plant growth. Dr. Jones explains that these cells, and the first set of primordial leaves are not connected directly to the vascular tissue, the plant’s transport system by which pathogens spread. Therefore, meristematic cells tend to be disease-free, whatever the condition of the mother. It takes a sharp blade, a dissecting microscope, and a lot of experience to learn, but as Dr. Jones explains, “harvesting explants from meristems is a routine micropropagation technique used by ‘Big Horticulture.’ One example is the strawberry. Viruses and pathogens are so prevalent that the strawberry industry must use meristematic culture to ensure pathogen free progeny.”

Epigenetics

Now let’s talk about epigenetics. We know that plants don’t have the option of physically moving away from stress or predation. Instead, they have evolved sophisticated ways of changing their own biology to adapt to and/or protect themselves. “Consider what happens to a mom exposed to a pathogen. The infected plant will start expressing (turning on) genes and making proteins that contribute to pathogen resistance,” says Dr. Jones. “These changes to gene expression are partly regulated by epigenetic modifications, chemical changes to DNA that increase or decrease the likelihood a cell will express a particular gene, but that do not actually modify the gene itself. Like annotations to a piece of music, epigenetic modifications don’t change the notes but rather how loud or soft, quickly or slowly the notes are played.”

There are more than 1,000 different viruses and mixed infections are very common

This is where it gets interesting. “Epigenetic modifications can be systemic and long lived. Plants infected by a pathogen or stressed by drought will present widespread epigenetic modifications to their DNA,” says Dr. Jones. “For an annual plant like cannabis, those modifications are relatively permanent. Thus a cutting from a mom having drought or pathogen adapted epigenetic programming will inherit that modified DNA and behave as if it were experiencing that stress, whether present or not.”

In the wild, this adaptability is critical for plant survival and reproduction, but to a grower, this is a less-than-ideal scenario. “The epigenetic modifications allowed the mother to tolerate the stress, which is great from the perspective of survival and fitness, but it comes at a cost. Some of the finite energy and resources that usually support plant growth and reproduction are instead channeled to stress response,” says Dr. Jones. This trade off results in reduction in overall plant yield and quality. “Those epigenetic changes result in a new phenotype for that mother,” says Dr. Jones. “All cuttings from her will reflect the new phenotype. This is one major mechanism underlying what many in the cannabis industry (incorrectly) call ‘genetic drift,’ or the loss of vigor over successive clonal generations.”

This is again where tissue culture can be such a game changer. The process of dedifferentiation, as explained in part 1 of this series, can rejuvenate a “tired” mother plant by inducing a kind of reboot– clearing accumulated epigenetic modifications that negatively impact progeny vigor and productivity. In the third part of this series, we will discuss the five stages of micropropagation, detailing the process of how you can grow plantlets in tissue culture. Stay tuned for more!

Judging a Craft Cannabis Competition

By Aaron G. Biros
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Willamette Week, a Portland-based publication, is hosting the 2017 Cultivation Classic with Farma, Cascadia Labs, Phylos Bioscience and the Resource Innovation Institute on May 12th. The event is a benefit for the Ethical Cannabis Alliance, an organization that promotes sustainability, labor standards and education surrounding the integrity and ethics of growing cannabis. Cultivation Classic is a competition for pesticide-free cannabis grown in Oregon, according to a press release.

Congressman Earl Blumenauer speaking at last year’s Cultivation Classic
Photo: Bridget Baker, 92bridges.com

While the event’s focus is on the competition, it is just as much a celebration of the craft cannabis community in Oregon. This year’s competition incorporates scientific collaboration like genetic sequencing for the winners by Phylos Bioscience and carbon accounting for all competitors. Keynote speakers include Ethan Russo, medical director of PHYTECS and Dr. Adie Po, co-founder of Habu Health. Congressman Earl Blumenauer, a prominent cannabis legalization advocate in Oregon, will also be speaking at the awards ceremony. You can check out the full schedule and speaker lineup here.

Raymond Bowser, breeder at Home Grown Natural Wonders, is a judge for this year’s Cultivation Classic. He speaks at cannabis conferences around the country and his business created a number of different strains, so he has experience with a myriad of growers and strains. “This time around everyone has really stepped up their game,” says Bowser. “The entries are noticeably better than last year.” When looking at the different samples sent to him, he sees a few key factors as most important in judging the quality. “What I am looking for is simple; a nice smell and a decent look, generally speaking,” says Bowser. “Aesthetics can tell you a lot about how it was grown, temperature changes and the overall care taken in cultivating and curing the flower.” For him, flavor, smell and aesthetics are the big variables to consider.

Photo: Bridget Baker, 92bridges.com

Those are factors that his company holds to high standards in their work, so he judges the samples based on the same variables. “It is what we strive for in our gardens and so far the samples I have tried are fantastic in that regard,” says Bowser. In other competitions that Bowser has judged in the past, they sent him between 40 and 60 strains to judge in seven days. “That is not conducive to a fair evaluation,” says Bowser. “Here, we are getting fourteen or so different strains, so we can sample one strain a day which is how I personally like to do it.”

Bowser is supportive of Cultivation Classic because of their emphasis on the craft industry. “We talk about craft cannabis and breeding craft cultivars at conferences around the country,” says Bowser. “With the rec industry growing so much, we see so many people cutting corners to save money, that it is refreshing to see growers take pride in the craft.” He also stresses the need for good lab testing and sound science in the trade. “I am big on lab testing; it is very important to get all the right analytics when creating strains,” says Bowser. “Cascadia is a solid choice for the competition; they have been a very good, consistent lab.” Emphasizing the local, sustainability-oriented culture surrounding the craft market, Bowser is pleased that this competition supports that same message. “We need to stay true to our Oregon roots and continue to be a clean, green, granola-eating state.”

Photo: Bridget Baker, 92bridges.com

Cascadia Labs is conducting the pesticide and cannabinoid analytics for all submissions and Phylos Bioscience will perform testing for the winners. According to Julie Austin, operations manager at Cascadia Labs, pesticide testing for the Oregon list of analytes was of course a requirement. “Some of the samples submitted had previous tests from us or from other accredited labs, but if they didn’t have those results we did offer a comprehensive pesticide test,” says Austin. The competition’s fee for submission includes the potency and terpenes analysis.

Jeremy Sackett, director of operations at Cascadia Labs, says they test for 11 cannabinoids and 21 terpenes. The samples are divided into groups of THC-dominant samples, CBD-dominant samples and samples with a 1:1 ratio of the two. “The actual potency data will be withheld from judges and competitors until the day of the event,” says Sackett. “We are data driven scientists, but this time we want to have a little fun and bring the heart of this competition back to the good old days: when quality cannabis was gauged by an experience of the senses, not the highest potency number.” The event will take place on May 12th at Revolution Hall in Portland, Oregon. Click here to get tickets to the event.