As part of our goal to become a cannabis ag-tech leader, at Segra we maintain a commitment to help educate the broader industry regarding our specific fields of expertise. In recent months we have been hearing from a growing number of operators that they've attempted plant tissue culture (TC) work within their own facilities and are struggling to achieve successful results or that they procured TC plants from other sources that were lack-luster. Confusion amongst growers regarding the various TC techniques is also common wherein some methods being touted by consultants actually have very low probability of producing reliable clean stock at all. In this edition of our Growth Notes newsletter, we will together explore the important topic of “how does a cultivator vet a potential TC partner?"
TC technologies are rapidly being adopted by top commercial cannabis growers globally with goals to both provide a reliable ongoing source of “pathogen free” clonal plants for production, and as a method to securely store Cannabis cultivars indefinitely for future production, breeding, or research purposes. However, Cannabis is a relatively new candidate for tissue culture technology. Related scientific literature and operational experience is fundamentally limited. Many emerging companies and consultants in this field offer products and services to growers. As an operator, how does one vet a potential TC technology provider? This decision is of utmost importance to the long-term success of your organization. We hope you find the question-and-answer dialog we have prepared below informative and useful.
What is the source of your TC expertise?
At Segra, our technologies and systems have been developed under the leadership of Dr. Sma Zobayed and his talented team of researchers. Dr. Zobayed, a leading specialist in tissue culturing with extensive international experience, has been granted three patents relating to TC (with another two patents pending) and has published over 50 scientific papers related to his research in the field. He earned a Ph.D. in plant tissue culture from the Department of Biological Sciences at the University of Hull (UK) and has won awards for his tissue culture work from the scientific community. Having worked on Cannabis in tissue culture for more than a decade, Dr. Zobayed possesses unique expertise in the field. Segra now has more than three years of direct hands-on experience including establishing well over 100 cultivars in vitro. In addition to Segra’s cultivar collection, we now manage cultivar libraries for more than 15 other producers and breeders at our lab in New Westminster, B.C.
What is your approach to pathogen testing? Which pathogens, how are they tested for, and how frequently?
All cultivars are routinely tested for the pathogens listed in the table below. Three consecutive negative results are required to validate any result. Currently, cultivars in storage are tested a minimum of twice a year, marketed cultivars are tested quarterly and each individual shipment is tested for Hop Latent Viroid. Certificates of Analysis to this effect are provided with every shipment.
For pathogen testing do you use internal tests, third-party tests, or both?
Segra uses both. We consider ourselves a leader in the development of cannabis pathogen assays, and routine testing is predominantly performed internally at our Genetics and Molecular Biology Laboratory. Segra utilizes third-party tests for additional testing and external validation in some scenarios. We are always happy to provide plant materials to other third-party labs at any customer’s request.
How frequently do you re-initiate cultivars?
Re-initiation is the process of bringing a cultivar out of the in vitro environment and then re-establishing it in vitro. This procedure is standard for most TC crops when re-initiation is completed every four to five years to ensure vigorous in vitro growth. Out of an abundance of caution we complete this process every two to three years.
What unique risks exist when implementing cannabis tissue culture and how do you manage them?
Somaclonal variation is a term used to describe genetic change that can occur in plantlets in the in vitro environment, resulting in a plantlet with a different genotype to the donor or source plant (reference genotype). Segra uses best practices during plantlet preservation and multiplication to mitigate the occurrence of somaclonal variation. We also routinely conduct DNA fingerprinting on our cultivars in tissue culture to monitor for the occurrence of somaclonal variation, if detected, individual plantlets or batches may be isolated to remove somaclonal variants.
Unsuccessful pathogen remediation:
Not all TC techniques provide the same efficacy in terms of pathogen remediation. Apical meristem tissue culture provides the highest probability of pathogen remediation wherein very small (< 1.0 mm) pieces of apical meristematic tissue are used to regenerate the cultivar. This tissue, while not connected to the vascular system of the plant, is essentially insulated from pathogens or organisms that may be present systemically. Segra always uses meristematic tissue culture in combination with other proprietary treatments that address virus and viroid contamination specifically. The regular pathogen testing we conduct(Table 1) also constantly monitors for these pathogens in our plant material ongoing.
Can you give some examples of your customers? How long have they been active clients?
We currently have over 50 customers we ship plantlets to in four different countries, and many of these accounts have been active for more than three years. We also maintain ongoing academic collaborations with institutions including Simon Frasier University and the University of British Columbia.
Do you use meristem, shoot tip, or nodal culture? What size is the tissue you extract from the source plant material?
Segra uses apical meristem as well as shoot tip culture. “Shoot tip”culture can offer a fast track to regenerate and resupply cultivars, provided that all post-initiation pathogen testing is successful. “Apical meristem” culture is a longer process but has a higher probability of pathogen remediation and is used for long term production and storage of all cultivars. At Segra, meristems are < 1.0 mm and shoot tips range from 1-5 mm. Nodal culture implies the use of plant nodes 2cm or larger. Segra does not use nodal culture as recent scientific literature calls this technique into question for successful pathogen remediation (Wei et. al., 2019).
How long does it take to regenerate a cultivar, and how many aseptic shoots do you store upon completion?
Cultivar regeneration through shoot tip culture takes approximately 4-6 months and meristem culture approximately 9-12 months. A minimum of 50 shootlets are always maintained in storage once the meristem process is finished. Click HERE to see a detailed description of our Regenerate and Preserve Program and to learn more about why we do both meristem and shoot tip initiations.
How much plant material do I need to provide for each cultivar I submit for TC Regeneration?
100-150 cuttings from 1-2 mother plants.
What data do you collect on your catalogue cultivars? Where is the data sourced?
Segra conducts cultivar characterization trials to collect cultivation data on all marketed cultivars before release, and we publish anonymized certificates of analysis from many of our customers on our website. Our data sets include, but are not limited to, plant growth characteristics, flower development, and cannabinoid/terpene analyses. Cultivar lineage and breeding information is also provided.
What pathogen testing is guaranteed for each shipment of plantlets?
A Certificate of Analysis is provided with each shipment, detailing the most recent pathogen testing (Table 1) of each cultivar. Additional Hop LatentViroid tests are conducted on the rooted plantlets prior to shipment, and test results are provided.
How do you ensure that I’m receiving the cultivar that I ordered?
Each outgoing shipment of plantlets is DNA fingerprinted and matched to the reference genotype. This ensures that each batch of plantlets shipped is a genetic match to the requested cultivar. Proof of this testing is also provided with shipment.
How are the plantlets rooted before shipping?
“Micro-Plug” plantlets are rooted in coco-peat propagation plugs before shipping. Bare-root plantlets are rooted in vitro and shipped free of media. Click HERE to learn more about our plantlet formats.
How are the plantlets packaged for shipping?
Micro-Plug plantlets are arranged in a fitted plastic tray, attached to a clear plastic vessel with lid. Bare-root plantlets are wrapped in sterile, moist tissue paper and packaged in plastic clamshell containers.
Describe your grading process when preparing shipments. Do you provide overage?
Plantlets are graded for quality and uniformity before shipping. We strive to provide the most consistent starting material to the customer possible with each shipment. Overages are always provided. Since we opened our doors at Segra our customers have achieved over 95% success rate regarding plant survival upon arrival.