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.

https://www.mdpi.com/2223-7747/10/1/185/pdf

The Past, Present and Future of Cannabis sativa Tissue Culture

For a long time, Cannabis sativa has been used for therapeutic and industrial purposes. Due to its increasing demand in medicine, recreation, and industry, there is a dire need to apply new biotechnological tools to introduce new genotypes with desirable traits and enhanced secondary metabolite production. Micropropagation, conservation, cell suspension culture, hairy root culture, polyploidy manipulation, and Agrobacterium-mediated gene transformation have been studied and used in cannabis. However, some obstacles such as the low rate of transgenic plant regeneration and low efficiency of secondary metabolite production in hairy root culture and cell suspension culture have restricted the application of these approaches in cannabis. In the current review, in vitro culture and genetic engineering methods in cannabis along with other promising techniques such as morphogenic genes, new computational approaches, clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR/Cas9-equipped Agrobacterium-mediated genome editing, and hairy root culture, that can help improve gene transformation and plant regeneration, as well as enhance secondary metabolite production, have been highlighted and discussed.

Advances and Perspectives in Tissue Culture and Genetic Engineering of Cannabis

In recent years high-THC (psychoactive) and low-THC (industrial hemp) type cannabis (Cannabis sativa L.) have gained immense attention in medical, food, and a plethora of other consumer product markets. Among the planting materials used for cultivation, tissue culture clones provide various advantages such as economies of scale, production of disease-free and true-to-type plants for reducing the risk of GMP-EuGMP level medical cannabis production, as well as the development and application of various technologies for genetic improvement. Various tissue culture methods have the potential application with cannabis for research, breeding, and novel trait development, as well as commercial mass propagation. Although tissue culture techniques for plant regeneration and micropropagation have been reported for different cannabis genotypes and explant sources, there are significant variations in the response of cultures and the morphogenic pathway. Methods for many high-yielding elite strains are still rudimentary, and protocols are not established. With a recent focus on sequencing and genomics in cannabis, genetic transformation systems are applied to medical cannabis and hemp for functional gene annotation via traditional and transient transformation methods to create novel phenotypes by gene expression modulation and to validate gene function. This review presents the current status of research focusing on different aspects of tissue culture, including micropropagation, transformation, and the regeneration of medicinal cannabis and industrial hemp transformants. Potential future tissue culture research strategies helping elite cannabis breeding and propagation are also presented.

/pdf/medical-cannabis-and-industrial-hemp-tissue-culture-present-1cz15oyotj.pdf

Medical Cannabis and Industrial Hemp Tissue Culture: Present Status and Future Potential.

Machine learning (ML) algorithms and artificial neural network for optimizing in vitro germination and growth indices of industrial hemp (Cannabis sativa L.)

Modeling and optimizing in vitro seed germination of industrial hemp (Cannabis sativa L.)

Existing Cannabis sativa micropropagation protocols use a limited number of cultivars and the results are often not reproducible. Currently, Murashige and Skoog basal salt mixture (MS) + 0.5 μmol/L...

DKW basal salts improve micropropagation and callogenesis compared with MS basal salts in multiple commercial cultivars of Cannabis sativa

Micropropagation of Cannabis sativa is an emerging area for germplasm storage and large-scale production of clean plants. Existing protocols use a limited number of genotypes and are often not reproducible. Previous studies reported MS + 0.5 μM TDZ to be optimal for Cannabis nodal micropropagation, yet our preliminary studies using nodal explants suggested this media may not be optimal. It resulted in excessive callus formation, hyperhydricity, low multiplication rates, and high mortality rates. Following an initial screen of four commonly used basal salt mixtures (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 μM TDZ and DKW + 0.5 μM TDZ were compared using five drug-type cultivars to determine if the preference for DKW was genotype-dependent. Four cultivars had significantly higher multiplication rates on DKW + 0.5 μM TDZ with the combined average being 1.5x higher than explants grown on MS + 0.5 μM TDZ. The canopy area was also significantly larger on DKW + 0.5 μM TDZ for four cultivars with the combined average being twice as large as the explants grown on MS + 0.5 μM TDZ. In the third experiment, callogenesis was compared using a range of 2,4-D concentrations (0-30 μM) on both MS and DKW and similarly, callus growth was superior on DKW. This study presents the largest comparison of basal salt compositions on the micropropagation of five commercially grown Cannabis cultivars to date.

/pdf/dkw-basal-salts-improve-micropropagation-and-callogenesis-1oifgy6vr6.pdf

DKW basal salts improve micropropagation and callogenesis compared to MS basal salts in multiple commercial cultivars of Cannabis sativa

Micropropagation of Cannabis sativa L. is an emerging area in tissue culture for genetic storage and large-scale production of clean planting material. However, existing protocols have been developed using a limited number of genotypes and are often not reproducible. Previous studies reported MS + 0.5 μM TDZ to be optimal for Cannabis micropropagation, yet in our preliminary studies this medium resulted in excessive callus formation, hyperhydricity, low multiplication rates, 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 μM TDZ and DKW + 0.5 μM 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 μM TDZ with the combined average being 1.5x higher than explants grown on MS + 0.5 μM TDZ. The canopy area was also significantly larger on DKW + 0.5 μM TDZ for four out of five genotypes with the combined average being twice as large as the explants grown on MS + 0.5 μM TDZ. In the third experiment, callogenesis was compared using a range of 2,4-D concentrations (0-30 μM) 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.

/pdf/basal-media-optimization-for-the-micropropagation-and-4romefkjm0.pdf

Basal media optimization for the micropropagation and callogenesis of Cannabis sativa L.

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The Past, Present and Future of Cannabis sativa Tissue Culture

DKW basal salts improve micropropagation and callogenesis compared with MS basal salts in multiple commercial cultivars of Cannabis sativa

DKW basal salts improve micropropagation and callogenesis compared to MS basal salts in multiple commercial cultivars of Cannabis sativa

Basal media optimization for the micropropagation and callogenesis of Cannabis sativa L.