White Paper Resources

Cannabis Tissue-Culture Propagation: Ensuring Genetic Consistency
Cannabis Tissue-Culture Propagation: Ensuring Genetic Consistency
Ulrich Reimann-Philipp, Ph.D. and Andrea L. Small-Howard, Ph.D.1
1GBSciences, Inc., Las Vegas, NV.
Abstract

Patients seeking medical relief through use of the plant Cannabis sativa need legal access to safe and effective products. While twenty-four states have passed legislation to allow patient access to medical cannabis, these state-regulated medical cannabis programs do not include comprehensive guidelines or monitoring agencies to ensure safety and consistency of the cannabis supplied in their state. In order for cannabis to become a true medical option, patients and their doctors need assurance that: 1) the cannabis is free from microbial contaminants and pesticides, and 2) the cannabis product has consistent levels of active ingredients for accurate dosing. Cannabis tissue propagation addresses both of these medical concerns, but it is only the beginning of a comprehensive process designed by GB Sciences for ensuring safety and consistency of cannabis production in the GrowBLOX™ technology suite. Tissue culture allows for non-sexual reproduction of desired cannabis genotypes in a sterile environment, and the potential to cryogenically bank the genotypes. The following report describes GB Sciences progress in adapting tissue culture methodologies for future commercial cannabis enterprises using their proprietary TissueBLOX system. .

Keywords: Cannabis sativa, tissue culture, plant propagation
Opportunities & Challenges in Cannabis Tissue Culture Propagation
Tissue-culture propagation has become a valuable tool for the propagation of many crop and ornamental plants. It is also a requirement for direct genetic modification through molecular biology techniques. However, the protocols differ widely and have to be adapted to each new species and cultivar and many have not been successfully propagated through tissue culture yet.

The legal restrictions of growing Cannabis has prevented extensive research on its tissue culture propagation. However, propagation and regeneration of Cannabis has been reported by a few research groups for a limited number of marijuana and industrial hemp cultivars. We have based our initial tissue culture trials on some of these published protocols1-3. As is usually the case in plant tissue culture, various modifications had to be made to adapt the protocols to the genotypes that GB Sciences is most interested in propagating. The modifications included changes to nutrient composition, agar and sucrose content, light conditions and grow media.
Tissue Culture Goals
GB Sciences has pursued propagation of its genetic resources through tissue culture with three main goals:
1.) Generate a large number of genetically identical offspring (clones) from a minimal number, preferably one highly performing plant, of “mother” plants. This minimizes gradual genetic change and variability that occurs when plants are propagated through multiple cycles of conventional cloning.

2.) Start each new growth cycle with plantlets that have passed through the sterile phase of tissue culture cultivation. The tissue culture phase eliminates any carryover of pests or pathogens and guarantees that each grow cycle starts with plantlets that are capable of maximizing their genetic potential.

3.) Establish a method for cryo-preservation of non-seed plant genetic material. By creating “artificial seeds” that can be indefinitely stored at ultra-low temperatures the genetic makeup of the original mother plant selected for its productivity and metabolite composition can be preserved and used to re-create the original plant even after many grow cycles.
Cannabis Tissue Culture Procedures
GB Sciences currently maintains a collection of 32 genotypes in the form of clones, seeds and tissue-culture explants. To date we have tested 6 genotypes in tissue culture propagation trials. We were able to regenerate shoots from axillary bud- and shoot tip explants from all of these genotypes. These shoots could be multiplied in-vitro with varying efficiency. Three of the genotypes, GBS0005, GBS0009 and GBS0030 could be regenerated and transplanted to either coco-coir media or aeroponic cultivation.
The most successful method for cannabis tissue propagation to date utilized the following steps:
1.) Explants: small (1 cm) segments of young stems with axillary buds or shoot tips of same size

2.) Sterilization: 20 minute wash in dilute sodium hypochlorite followed by multiple washed in sterile distilled water

3.) Shoot-Induction and Multiplication: cultivation of the explants on agar-solidified MS or DKW media supplemented with different auxin growth regulators (depending on the genotype) for 2-3 weeks. Micro-shoots and buds from these cultures were also used as sterile starting material for new shoot induction and multiplication.

4.) Shoot Elongation: Transfer of individual shoots to agar-solidified growth media supplemented with auxins, alone or in combination with gibberellins and cytokinins (again depending on the genotype) for 2-4 weeks.

5.) Root Induction: Transfer of elongated shoots to either agar-solidified media or sterile rockwool-plugs supplemented with auxins (IBA or NAA) for 3-4 weeks.

6.) Transfer out of tissue culture: rooted plantlets in rockwool plugs were directly transplanted to coco-coir grow media for conventional grow or to baskets with expanded clay pebbles for aeroponic cultivation. The roots from plantlets grown on agar-media were first thoroughly washed and then transplanted in the same way.
Experimental Results
Optimal results were obtained with explants from genotypes GBS0005, GBS0009 and GBS0030. These yielded 2-5 shoots per explant and could be regenerated to complete plantlets that adapted readily to aeroponic or conventional grow. The following image series documents the typical growth stages:
Next Steps
GB Sciences envisions that a full understanding of the metabolomes of individual Cannabis cultivars will (a) address the paradox that separates the therapeutic experiences of countless cannabis users and the medical understanding of cannabis as therapy, and (b) bring Cannabis therapeutics into the era of personalized, precision medicine, where patients are matched with strains based on a defined relationship between metabolome and efficacy.

How variant are the extant strains of Cannabis? The first large scale metabolomics attempt to differentiate Cannabis cultivars using metabolomes was published in 2004 
GB Sciences will be utilizing tissue-culture propagation regulated by the growth control software and components developed for the TissueBLOX plant propagation system. Cannabis tissue-culture propagation allows for the mass-production of genetically identical, sterile plantlets and materials for the cryo-preservation of genetic assets. These steps are the beginning of the comprehensive process designed by GB Sciences for ensuring safety and consistency of cannabis production in the GrowBLOX™ technology suite. Genetic consistency as well as control of the critical growth parameters during the later stages of the plant growth cycle are essential in order to ensure safety and consistency within medical cannabis products.
Konan et al. (1997): An Efficient Mass Propagation System for Cassava (Manihot esculenta Crantz) Based on Nodal Explants and Axillary Bud-Derived Meristems. Plant Cell Reports 16: 444–449

Lata et al. (2009): Thidiazuron-Induced High-Frequency Direct Shoot Organogenesis of Cannabis Sativa L. In Vitro Cell.Dev.Biol.-Plant 45:12–19

Wang et al. (2009): A Micropropagation System for Cloning of Hemp (Cannabis sativa L.) by Shoot Tip Culture. Pak. J. Bot., 41(2): 603-608

Lata et al. (2011): Molecular Analysis of Genetic Fidelity in Cannabis sativa L. Plants Grown from Synthetic (Encapsulated) Seeds Following In Vitro Storage. Springer Biotechnol. Lett. Online

Lata et al. (2010): High Frequency Plant Regeneration from Leaf Derived Callus of High Δ9-Tetrahydrocannabinol Yielding Cannabis sativa L. Planta Med 76: 1629–1633

Lusarkiewicz-Jarzina et al (2005): Influence of Cultivar, Explant Source and Plant Growth Regulator on Callus Induction and Plant Regeneration of Cannabis Sativa L. Acta Biologica Cracoviensia Series Botanica 47/2: 145–151

Lata et al. (2012): In Vitro Germplasm Conservation of High A9 –Tetrahydrocannabinol Yielding Elite Clones Of Cannabissativa L. under Slow Growth Conditions. Acta Physiol Plant 34:743--750