An Exclusive Look at Cannabis Tissue Culture, a Transformative Technology – Part 1
July 18, 2016 at 4:37 pm

Why Tissue Culture is Key to the Future of the Cannabis Industry
Part I: An Introduction to Cannabis T.C.

Guest post by Michael Stevens, Founding Partner at Meristematic Labs

Over the past decade, the scale and sophistication of the Cannabis Industry in the United States has developed dramatically. This aggressive transition from isolated small level cultivators to full-scale industrial horticulture has bought about a new set of problems for both government regulators and the cannabis business community. These problems have become new opportunities for innovative companies looking to push the limits of what is possible with cannabis cultivation. One of the most significant, changes coming to the cannabis industry is the application and use of Plant Tissue Culture Technologies.

What exactly is Plant Tissue Culture? The term, is usually defined as the controlled aseptic culturing of whole plants, plant cells, tissues, organs and protoplasts. Aseptic meaning, free from contaminates like insects and other external microorganisms. The procedure for introducing plant cells into culture is relatively simple.

A small section of the plant called the explant, is cleaned and placed in to a sterilized vessel containing a media substrate of agar, sugars and growth regulating chemicals. In the case of Cannabis, micro nodes are taken from the original stock plant and placed in sterilized test tubes to grow. After 10-14
days the node has developed into a juvenile cannabis plantlet, with no roots. Given the proper conditions and fresh growth media, a plantlet can be indefinitely divided into other explants for new cultures. These new cultures can then be moved into a cannabis depository for storage, placed into bioreactors, used for experimental trials or transferred into rooting media & acclimated for cultivation in the greenhouse environment. The process of tissue culturing is generally described in four distinct stages.

Stage #1 Establishment of Aseptic Culture
Stage #2 Multiplication
Stage #3 In-vitro Rooting
Stage #4 Acclimation

Tissue culture was first used on a larger scale by the orchid industry in the early 1950’s. The technology proved to be useful for propagating orchids with seed stock that is difficult to germinate. Today, Plant Tissue Culture technologies are used to preserve endangered species like the Ghost Orchid (Dendrophylax lindenii). Work done by Dr. Michael Kane & Dr. Hoang Nguyen at the University of Florida[i] is using tissue culture to grow Ghost Orchids in the lab. Once fully grown the orchids are reintroduced back to their native habitat in the Florida Panther National Wildlife Refuge.

The culturing of plant cells in-vitro is the first step in a number of specialized agri-industrial and biotechnological processes. Living cultures can be use for micro-propagation, haploid production, somatic embryo-genesis, synthetic seed production callus cell production, protoplast isolation and much more. The early stages of plant cryopreservation uses tissue cultured cells & special sugars to store the living cells at -190ºC. Micro-propagation uses tissue-cultured cells grown in bioreactors to produce large numbers of identical plant shoots for starter plants. Specialized plant cells like anther, callus and polyploidy cells can be produced and isolated using Plant Tissue Culture. It is simply a matter of time until these types of high-level agri-technologies are applied to the cannabis industry.

Only in the past few years have cultivators and VCs taken cannabis tissue culture seriously. As real demand increases for cannabis and cannabis related products, cultivators of all sizes will need large quantities of clean, healthy, true to type starter plants. Intellectual property based around cannabis phenotype development and developed plant traits will rely heavily on tissue culture and cryopreservation as methods of possession of phenotypes. One of the safest ways for medically significant cannabis phenotypes to be transported and exchanged is as tissue culture in sealed vessels. Both government regulators and cannabis industry professionals need to be educated about the benefits of this technology so it can continue to develop.

[i] University of Florida
UF/IFAS scientists preserve the endangered Ghost Orchid – January 26, 2016
https://news.ifas.ufl.edu/2016/01/ufifas-scientists-preserve-the-endange...

About the author:
Michael J. Stevens has been an innovator in the cannabis community for the last 20+ years. As a cannabis activist living in Southern California in the mid 90s, he participated in a number of local direct actions to protest cannabis prohibition. Eventually relocating to San Francisco in 1997, he quickly integrated into the Bay Area cannabis community. During his time in Northern California he developed a number of popular cannabis phenotypes.

In 2009, Mr. Stevens started working on the development of an industrial process for the Micro-propagation of Cannabis plants. January 2015, Meristematic Labs was launched.

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Segra Biogen and Chong’s Choice to Develop Industrial Scale Cannabis Tissue Culture Facility
June 27, 2016 at 9:47 am

Evergreen, Licensor of Tommy Chong’s Marijuana Brand “Chong’s Choice” and Segra Form Strategic Alliance That Brings True Hi Tech to the $25 Billion Cannabis Industry
“Joint” Press Release

LOS ANGELES, CA, Jun 27, 2016 – Segra Biogen Corp., a Cannabis Science Company, and Evergreen Licensing, LLC, a California Corporation and the licensor of Tommy Chong’s brand “Chong’s Choice,” have announced plans for Segra to become the science advisor for the Chong’s Choice brand.

Segra will advise Chong’s Choice on matters of industrial scale Cannabis tissue culture and genetics, as well as the design, construction, and operation of clean growing facilities.

Further, Segra Biogen Corp and Chong’s Choice will develop and operate an industrial scale Cannabis tissue culture facility in California.

Segra will also operate a molecular biology laboratory for the purpose of genotyping – genetically identifying, researching, and indexing (“fingerprinting”) – a broad variety of commercially available Cannabis strains.

Genotyping both protects Chong’s Choice products against genetic drift, and establishes a platform for the development of new, high value, proprietary strains and ancillary products.

Purity and quality are central to the widely known and highly respected Chong’s Choice brand. Now they are incorporating next generation Cannabis technology into their production.

Selecting the Segra Biogen scientific team and implementing its Quality Management System, Chong’s Choice confirms its long-term commitment to producing the purest and highest quality Cannabis products in the marketplace.

Brian Vecchio, Evergreen Co-Founder states: ”Evergreen is investing in high technology for the future; we want the cannabis brands we represent to be the industry standard for quality. With Segra on our team, we are well on our way to becoming the number one brand worldwide.”

Segra looks forward to working with the Chong’s Choice team; their deep commitment to remaining the number one brand based on product quality, is simply part of their DNA.

ABOUT SEGRA BIOGENESIS CORP:

Segra Biogenesis Corp. is a Cannabis Science Company focused on standardizing cannabis production to ensure consistent and repeatable quality. Our team is driven by multidisciplinary experience in medicine, genetics, cultivation techniques, natural medicines, biochemistry, agriculture, and information technology. Segra’s goal is to research, develop, test, produce, and market products that are effective, safe and have the highest quality for promoting and maintaining good health.

The Segra team has built its Quality System to address the most complex and highest regulated market in the world: Canada. The Segra Quality Management System, grow room design, tissue culture and genotyping, position the Company as a leading advisor in Quality.

ABOUT CHONG’S CHOICE:

The name Tommy Chong is thoroughly identified with Cannabis. As one-half of the legendary comedy duo Cheech & Chong, he helped to pioneer Stoner Comedy, a subversive style that came to symbolize counter-culture entertainment in the 1970’s. At the heart of this brand of entertainment was, of course, cannabis.

On screen and off, Tommy became a vocal advocate for his favorite herb. In a time when cannabis had to be talked about in whispers, Tommy was a loud voice for the movement, working tirelessly to de-stigmatize the use of this healing plant. Now that society has moved in a more positive direction regarding the medicinal and recreational use of cannabis, Tommy’s voice continues to ring out loudly, touting its benefits and rightful place in society.

CONTACT INFORMATION

Segra Biogenesis Corporation
223 – 21300 Gordon Way
Richmond B.C. V6W IM2
Canada
todd@segrabiogen.com
www.segrabiogen.com

Evergreen Licensing LLC
3218 Glendale Blvd.
Los Angeles California 90039
United States
brian@chongschoice.com
www.chongschoice.com

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Tissue Culture for Cannabis Cultivation Could Replace Cloning as the Gold Standard
June 3, 2016 at 9:45 am

Applications and Advantages of Tissue Culture in Cannabis Cultivation
Large scale agriculture is at its most efficient when it has a reliable, uniform supply of juvenile plantlets to grow from. Cannabis culture is no different, and starting vegetation phase of growth from already hardened plantlets (as opposed to seeds) saves time and resources otherwise spent on attempting to propagate non-viable or substandard / undesirable seeds within a group. An additional advantage in regulated environments is that the entirety of permitted canopy space can be devoted to growing plants instead of maintaining vegetative mothers and clones.

Clonal (that is, genetically identical) plantlets – derived either from mother plant cuttings, or other methods as discussed below – add the even more important advantage of maintaining uniform strain genetics, leading to predictable growth behaviour and mature plant characteristics such as THC and CBD content, terpene profiles, and other strain specific traits which are associated with a strain name and any downstream product branding. Also, clonal plants all reach milestone stages within a much narrower window than plants derived from seeds. Seeds, in contrast, are by their very biological nature genetic reassortants of their parental stocks, making each seed unique; useful for long term selective breeding programs where variety to select from is of use, but not at all desirable once a lineage is selected and uniform product is desired.

Cannabis cultivators have of course known this for a long time. While seeds are used for some smaller scale growth applications, established growth operations almost all work via clonal mother plant cuttings. This ensures product uniformity, but is relatively labor intensive and requires significant physical space for the cutting and hardening stages. Mother plants have a finite lifespan and limited number of cuttings they may provide, making the method relatively slow to scale up. Finally, traditional mother and cut clone systems are susceptible to all of the same plant pathogens (such as Fusarium spp. or powdery mildew) that can infect vegetating and flowering cannabis crops. Infestation of mother plants and clones can in worst case scenarios lead to the complete loss of strain stock – a serious risk for the grower.

A technique widely used in other branches of industrial scale agriculture has the potential to address all of these shortcomings of classical mother and cut clones, and do so economically and with vast scalability. As you’ve probably guessed from the title, this approach is tissue culture (“TC”). Tissue culture from plants involves taking a small amount of plant tissue which is induced to return to- and maintained in- a primitive stage (in the form of ‘calluses’). These cells can be propagated indefinitely in defined synthetic media; when plantlets are desired, some of these cells are taken out and then induced to differentiate into all of the different tissue that makes up a complete plant.

The first benefit from the method is that these cells can be maintained in large quantities in very small volumes and can be expanded to hundreds of thousands of cells very quickly, and again, in very small volumes. The second benefit is in scalability; hundreds of thousands of plantlets can be induced in far less time and effort than creating mere hundreds of cuttings. A crucial third benefit of TC over traditional cloning methods is that by stringent control of the maintenance conditions, appropriate growth media additives, and proper handling techniques combined with good facility sanitation practices, strains can be maintained and propagated free of detectable pathogens (TC methods require aseptic handling and the presence of pathogens are readily detected and eliminated).

So now we appreciate that TC can generate vast numbers of identical, healthy “calluses”; how do we go about turning these into plants that we recognize as plants, suitable to move forward to vegetation and flowering stages?

The process generally involves taking callus stock, expanding the number of cells in liquid media as single cells, then plating individual cells in their own culture chambers, then add the appropriate growth factors and hormones that induce them to differentiate into all of the normal tissue types that make up complete plants; as the cells divide they will differentiate into roots, stems, and leaves. Once this differentiation proceeds, they grow into complete plantlets. When these plantlets reach around the 6-8” tall stage, these are then ready to pass on for handling as normal juvenile plants, without any differences in process for subsequent growth.

An added benefit of TC processes is that calluses can be maintained long term without differentiation, at very low cost and low space requirements. Particularly for an operation wanting to maintain multiple strain types for long periods, but able to bring them into production quickly and in large numbers, TC is an economical and reliable solution.

In summary, TC is a well-established method for the retention, clonal expansion, and on-demand production of vast numbers of intrinsically healthy, uniform juvenile plants while offering cost and space savings over traditional methods of any appreciable scale. TC methods are the industry standard for a wide range of agricultural or ornamental plant species – from blueberries to strawberries to birch trees – and going forward as cannabis production becomes mainstream industry and requires the associated scale and uniformity, TC methods will become the obvious method of choice for forward-thinking producers. Whether that comes via development of in-house expertise and infrastructure, or through contracting services out to established TC nurseries, is a matter of choice; but one or the other will surely become the gold standard for cannabis stock maintenance in the very near future.

About the authors:

Dr. John Brunstein is the Chief Scientific Officer and Dr. Kevin She is the Quality Assurance Manager/Senior Scientist for Segra Biogenesis Corp. Segra is a cannabis sciences company focused on the standardization of cannabis products through the use of its Quality Management System, genotyping, and tissue culture.

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