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The recent legalization of Cannabis sativa L. in many regions has revealed a need for effective propagation and biotechnologies for the species. Micropropagation affords researchers and producers methods to rapidly propagate insect-/disease-/virus-free clonal plants and store germplasm and forms the basis for other biotechnologies. Despite this need, research in the area is limited due to the long history of prohibitions and restrictions. Existing literature has multiple limitations: many publications use hemp as a proxy for drug-type Cannabis when it is well established that there is significant genotype specificity; studies using drug-type cultivars are predominantly optimized using a single cultivar; most protocols have not been replicated by independent groups, and some attempts demonstrate a lack of reproducibility across genotypes. Due to culture decline and other problems, the multiplication phase of micropropagation (Stage 2) has not been fully developed in many reports. This review will provide a brief background on the history and botany of Cannabis as well as a comprehensive and critical summary of Cannabis tissue culture. Special attention will be paid to current challenges faced by researchers, the limitations of existing Cannabis micropropagation studies, and recent developments and future directions of Cannabis tissue culture technologies.

Cannabis sativa is usually clonally propagated from plants in the vegetative phase. However, phenotypic traits such as yield and chemical composition can only be assessed in unfertilized plants reaching the end of their life cycle and there are no peer-reviewed methods to propagate flowering plants. In this study, immature (three cultivars) and mature (one cultivar) floral explants were cultured on thidiazuron and shoot development was observed in both the immature and mature explants. This provides the first report of micropropagation from floral tissues in C. sativa and will enable plants to be clonally propagated up to the date of harvest.

Oxidative browning is a common and often severe problem in plant tissue culture systems caused by the accumulation and oxidation of phenolic compounds. The current study was conducted to investigate a novel preventative approach to address this problem by inhibiting the activity of the phenylalanine ammonia lyase enzyme (PAL), thereby reducing the biosynthesis of phenolic compounds. This was accomplished by incorporating 2-aminoindane-2-phosphonic acid (AIP), a competitive PAL inhibitor, into culture media of Artemisia annua as a model system. Addition of AIP into culture media resulted in significant reductions in visual tissue browning, a reduction in total phenol content, as well as absorbance and autoflourescence of tissue extracts. Reduced tissue browning was accompanied with a significant increase in growth on cytokinin based medium. Microscopic observations demonstrated that phenolic compounds accumulated in discrete cells and that these cells were more prevalent in brown tissue. These cells were highly plasmolyzed and often ruptured during examination, demonstrating a mechanism in which phenolics are released into media in this system. These data indicate that inhibiting phenylpropanoid biosynthesis with AIP is an effective approach to reduce tissue browning in A. annua. Additional experiments with Ulmus americana and Acer saccharum indicate this approach is effective in many species and it could have a wide application in systems where oxidative browning restricts the development of biotechnologies.

Micropropagation of Cannabis sativa L. is an emerging area of research for genetic storage and large-scale production of clean planting material. However, existing protocols were developed using a limited number of genotypes and are often not reproducible. Previous studies reported MS + 0.5 μ M TDZ to be optimal for Cannabis micropropagation, yet in our preliminary studies this medium resulted in excessive callus formation, hyperhydricity, low multiplication, and high mortality rates. Following an initial screen of five basal salt mixtures commonly used for micropropagation (WPM, MS, B5, BABI, and DKW), we determined that DKW produced the healthiest plants. In a second experiment, the multiplication rate and canopy area of explants grown on MS + 0.5 μ M TDZ and DKW + 0.5 μ M TDZ were compared using five drug-type cultivars to determine if the preference for DKW was genotype-dependent. Four out of five genotypes had significantly higher multiplication rates on DKW + 0.5 μ M TDZ with the combined average being 1.5x higher than explants grown on MS + 0.5 μ M TDZ. The canopy area was also significantly larger for plants cultured on DKW + 0.5 μ M TDZ for four out of five genotypes with the combined average being twice that of explants grown on MS + 0.5 μ M TDZ. In the third experiment, callogenesis was compared using a range of 2,4-D concentrations (0-30 μ M) on both MS and DKW. Greater callus growth was observed on DKW than on MS. While further improvements are likely possible through media optimization, this study represents an important step towards developing standardized micropropagation practices for Cannabis.

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BackgroundDue to the complex process of designing and manufacturing new plant tissue culture vessels through conventional means there have been limited efforts to innovate improved designs. Further, development and availability of low cost, energy efficient LEDs of various spectra has made it a promising light source for plant growth in controlled environments. However, direct replacement of conventional lighting sources with LEDs does not address problems with uniformity, spectral control, or the challenges in conducting statistically valid experiments to assess the effects of light. Prototyping using 3D printing and LED based light sources could help overcome these limitations and lead to improved culture systems.ResultsA modular culture vessel design in which the fluence rate and spectrum of light are independently controlled was designed, prototyped using 3D printing, and evaluated for plant growth. This design is compatible with semi-solid and liquid based culture systems. Observations on morphology, chlorophyll content, and chlorophyll fluorescence based stress parameters from in vitro plants cultured under different light spectra with similar overall fluence rate indicated different responses in Nicotiana tabacum and Artemisia annua plantlets. This experiment validates the utility of 3D printing to design and test functional vessels and demonstrated that optimal light spectra for in vitro plant growth is species-specific.Conclusions3D printing was successfully used to prototype novel culture vessels with independently controlled variable fluence rate/spectra LED lighting. This system addresses several limitations associated with current lighting systems, providing more uniform lighting and allowing proper replication/randomization for experimental plant biology while increasing energy efficiency. A complete procedure including the design and prototyping of a culture vessel using 3D printing, commercial scale injection molding of the prototype, and conducting a properly replicated experiment are discussed. This open source design has the scope for further improvement and adaptation and demonstrates the power of 3D printing to improve the design of culture systems.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-017-0156-8) contains supplementary material, which is available to authorized users.