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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.

Time & Resources

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.

Applications for Tissue Culture in Cannabis Growing: Part 1

By Aaron G. Biros
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Dr. Hope Jones, chief scientific officer of C4 Laboratories, believes there are a number of opportunities for cannabis growers to scale their cultivation up with micropropagation. In her presentation at the CannaGrow conference recently, Dr. Jones discussed the applications and advantages of tissue culture techniques in cannabis growing.

Dr. Hope Jones, chief scientific officer at C4 Labs

Dr. Jones’ work in large-scale plant production led her to the University of Arizona Controlled Environment Agriculture Center (CEAC) where she worked to propagate a particularly difficult plant to grow- a native orchid species- using tissue culture techniques. With that experience in tissue culture, hydroponics and controlled environments, she took a position at the Kennedy Space Center working for NASA where she developed technologies and protocols to grow crops for space missions. “I started with strawberry TC [tissue culture], because of the shelf life & weight compared with potted plants, plus you can’t really ‘water’ plants in space- at least not in the traditional way,” says Dr. Jones. “Strawberries pack a lot of antioxidants. Foods high in antioxidants, I argued, could boost internal protection of astronauts from high levels of cosmic radiation that they are exposed to in space.” That research led to a focus on cancer biology and a Ph.D. in molecular & cellular biology and plant sciences, culminating in her introduction to the cannabis industry and now with C4 Labs in Arizona.

Working with tissue culture since 2003, Dr. Jones is familiar with this technology that is fairly new to cannabis, but has been around for decades now and is widely used in the horticulture industry today. For example, Phytelligence is an agricultural biotechnology company using genetic analysis and tissue culture to help food crop growers increase speed to harvest, screen for diseases, store genetic material and secure intellectual property. “Big horticulture does this very well,” says Dr. Jones. “There are many companies generating millions of clones per year.” The Department of Plant Sciences Pomology Program at the Davis campus of the University of California uses tissue culture with the Foundation Plant Services (FPS) to eliminate viruses and pathogens, while breeding unique cultivars of strawberries.

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

First, let’s define some terms. Tissue culture is a propagation tool where the cultivator would grow tissue or cells outside of the plant itself, commonly referred to as micropropagation. “Micropropagation produces new plants via the cloning of plant tissue samples on a very small scale, and I mean very small,” says Dr. Jones. “While the tissue used in micropropagation is small, the scale of production can be huge.” Micropropagation allows a cultivator to grow a clone from just a leaf, bud, root segment or even just a few cells collected from a mother plant, according to Dr. Jones.

The science behind growing plants from just a few cells relies on a characteristic of plant cells called totipotency. “Totipotency refers to a cell’s ability to divide and differentiate, eventually regenerating a whole new organism,” says Dr. Jones. “Plant cells are unique in that fully differentiated, specialized cells can be induced to dedifferentiate, reverting back to a ‘stem cell’-like state, capable of developing into any cell type.”

Cannabis growers already utilize the properties of totipotency in cloning, according to Dr. Jones. “When cloning from a mother plant, stem cuttings are taken from the mother, dipped into rooting hormone and two to five days later healthy roots show up,” says Dr. Jones. “That stem tissue dedifferentiates and specializes into new root cells. In this case, we humans helped the process of totipotency and dedifferentiation along using a rooting hormone to ‘steer’ the type of growth needed.” Dr. Jones is helping cannabis growers use tissue culture as a new way to generate clones, instead of or in addition to using mother plants.

With cannabis micropropagation, the same principles still apply, just on a much smaller scale and with greater precision. “In this case, very small tissue samples (called explants) are sterilized and placed into specialized media vessels containing food, nutrients, and hormones,” says Dr. Jones. “Just like with cuttings, the hormones in the TC media induce specific types of growth over time, helping to steer explant growth to form all the organs necessary to regenerate a whole new plant.”

Having existed for decades, but still so new to cannabis, tissue culture is an effective propagation tool for advanced breeders or growers looking to scale up. In the next part of this series, we will discuss some of issues with mother plants and advantages of tissue culture to consider. In Part 2 we will delve into topics like sterility, genetic reboot, viral infection and pathogen protection.

Cannabis, Soil Science and Sustainability Part II: The ‘Roots’ of Sustainable Cultivation

By Drew Plebani
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The modern chemical agricultural approach is based on the assumption that chemical science has discovered all facets of plant nutritional requirements. It is clear that the traditional NPK approach to plant/soil systems has its limitations, both from an ecological perspective and in terms of its ability to create nutrient-dense food.

Soil and plant systems have existed together for millions of years and have evolved the capacity to coexist in a way that is mutually beneficial. Plants have been fed and evolved with these biological and environmental stimuli over millennia.

Looking to the geologic record for evidence, we can see that it shows that invertebrates, fungi and early vascular plants appeared on land roughly 400 million years ago, the first seed bearing plants about 360 million years ago and the first flowering plants 130 million years ago. What does this mean? The soil food web has been in existence for millions of years and significant evidence exists that plants and soil biology have co-evolved together for millennia.

geologictimescale
The Geologic Time Scale

Between mineral rich soils and the soil food web, this natural system has been able to create and provide significant plant available nutrients, certainly enough to facilitate the successful life cycle of many species. Clearly from an evolutionary context this system has been able to facilitate maximum genetic expression and the ongoing evolution of biologic species.

In the not-too-distant past, agricultural fertilization practices were based on the existence of a diversity of plant and animal byproducts, animal manures, green manures, etc. These were reintroduced to the system and combined with the appropriate biologic populations, resulting in the decomposition of these organic material inputs and their conversion into plant-available nutrients.

An overview of traditional farming practices provides substantial evidence that farming has been occurring for at least 10,000 years. Why, with such a long history of symbiotic interactions between biologic species, are we now witnessing the mass deterioration of arable land, and agricultural commodities containing lower nutritional value?

Mycelium, the vegetative part of a fungus bacteria colony, seen breaking through rock.
Together, indigenous mycelium and plant roots seen turning rock into soil

An interesting common question among the conventional farming community, when the topic of organics or sustainability comes up, is “how are you going to feed the world?” Well that goal certainly will not be well served by the development of shelf stable, but low nutrient-dense foods. A greater volume of low nutrient-value foods certainly does not seem like a winning approach. Supporting agricultural systems that encourage the development of sustainable systems via locally produced, nutrient-dense food is a good start.

And the same holds true for cannabis. In fact, the parallels between the production of high quality nutrient dense foods and high quality cannabis products are quite significant.

Nutrient density in crops results from balanced, mineral rich soils, and a diversity of organic materials and biologic life, these elements provide the framework to facilitate the creation of a highly functional, biologic nutrient cycling system. A highly functional soil system results in more nutrient-dense crops, which contain measurably larger quantities of different phytonutrients, vitamins, minerals, flavonoids, and terpenes as compared to a system operating at a lower level of biologic efficiency.

commercialcultivator
Nutrient-dense cannabis flowers

Benefits that have been observed from nutrient-dense crops are: more pest and disease resistance in the vegetative and fruiting stages, greater yield, more complex and intense flavors and a longer shelf life.

Ultimately advancement in any cultivation system means finding and defining limiting factors in the given system. The objective should be ensuring the maximum biologic vitality of the components of said system and its outputs. Practically speaking, in order to enable the full genetic potential of biologic species, this means identifying and working toward the removal of limiting factors. Minimizing or entirely alleviating the factors that limit maximum plant growth will undoubtedly net positive gains and must be an integral component to any sustainable cultivation strategy.

commcultivator3
Cannabis growing in a polyculture

The Earth has provided us with a highly successful, multi-million-year-old biologic system, capable of providing abundant plant available nutrients on demand, a dynamic which must be integral to appropriate and intelligent systems design.

In the pursuit of sustainability, perhaps it is time to return to our roots and begin to pursue dynamics that are mutually beneficial to all forms of biologic life.

In the next article, we will take a step back from viewing sustainability through the lens of soil and plant specific cultivation methodologies, and focus on the broader context of sustainability in cultivation systems. We will look at sustainability from the context of operational efficiency, and provide a case study from a 400-light commercial indoor cannabis operation. The case study will provide evidence that, in order to achieve higher levels of sustainability, both cultivation strategies and operational efficiency must be factored into the equation. As we will see, true sustainability is created through the efficient design, incorporation, use and management of system elements, all of which can, when appropriately designed, work together to create improved efficiency for the system.

Researching Cannabis Genetics: A Q&A with CJ Schwartz, Ph.D.

By Aaron G. Biros
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Studying cannabis genetics is a convoluted issue. Strain classification, medicinal effects and plant breeding are particular areas in the science of cannabis that still require heavy research. Marigene, a company researching cannabis genetics, is currently working with universities and research institutes to help map the cannabis genome and catalog genetic variation.

cjschwartzmarigene
CJ Schwartz, Ph.D.

According to CJ Schwartz, Ph.D., chief executive officer and founder of Marigene, their mission is to “to classify, certify, and improve cannabis.” After studying genetics and cellular biology at the University of Minnesota, Schwartz received his Ph.D. in biochemistry from the University of Wisconsin. His research in the past decade has focused on genetic variations that control flowering time, discovering the expression of a gene called Flowering Locus T leads to differential flowering time of plants and is dependent on their native locations. We sat down with Schwartz to learn more about his research and collaborative efforts.


Cannabis Industry Journal: Why are you researching mapping the cannabis genome?

CJ Schwartz, Ph.D: We seek to identify the genetic differences among cannabis strains and the genes responsible for these differences. Genetic differences are what cause different strains to have different effects. DNA allows reproducibility, consistency, and transparency for your cannabis strains.

The more information we gather about cannabis genetics, the more tools we have available to create tailored strains. Cannabis is a targeted compound. It interacts with a very specific system in the human body, similar to hormones, such as insulin. Understanding the cannabis genome will help bring legitimacy and integrity to cannabis products, and allow us to better understand how chemicals from cannabis interact with the human brain. Genetic identification can provide a method of certification to more comprehensively describe plant material.

Schwartz doing sample preparation on the lab bench.
Schwartz doing sample preparation on the lab bench.

CIJ: How did you get involved in cannabis research?

Schwartz: My interest in cannabis guided my research career. Cannabis may not be a cure-all, but it has significant and measurable medicinal effects for many patients.

To allow true development of cannabis products, we need more science! Our genetic analysis is required for normalization and acceptance of cannabis products, but also essential for future breeding efforts to develop better and more useful plants.

Our sister company, Hempgene, is applying all of the same technology and techniques for hemp research. One focus of Hempgene is to manipulate flowering time in select hemp cultivars so that they mature at the appropriate time in different environments.

CIJ: What do you hope to accomplish with your research?

Schwartz: We can develop or stabilize a plant that produces a very specific chemical profile for a specific condition, such as seizures, nausea or pain. By breeding plants tailored to a patient’s specific ailment, a patient can receive exactly the medicine that they need and minimize negative side effects.

The current term describing the interaction of cannabis compounds is called the entourage effect. Interactions among compounds can be additive or synergistic. The entourage effect describes synergistic effects, where small amounts of compound A (e.g. Myrcene) vastly increase the effects of compound B (e.g. THC). Instead of flooding one’s body with an excessive amount of chemicals to get a non-specific effect, cannabis plants can be bred to produce a very specific effect.

labmarigene
A view of some of the work stations inside the laboratory at Marigene.

Currently our goal is to catalog the natural genetic variation of cannabis, and to identify DNA changes that affect a trait of interest. Once superior variants of a gene are identified, those variants can be combined, by marker-assisted breeding, to produce new combinations of genes. How different cannabis chemicals interact to produce a desired effect, and how different human genetics influence the efficacy of those chemicals should be the ultimate goal of medical marijuana research.

We are working closely with academic institutions and chemical testing labs to gather data for establishing correlations between specific cannabis strains and desirable chemical profiles. Our closest collaborator, Dr. Nolan Kane at UC-Boulder, is working to complete the Cannabis genomic sequence and generate the first high- resolution cannabis genetic map.

We are currently accepting samples and we produce a report in roughly two to three months. For one sequencing run, we identify 125 million pieces of DNA that are 100 base pairs long. We get so much information so there is a considerable time commitment.