Recent advances in cannabis biotechnology

Cultivation of cannabis plants (Cannabis sativa L., marijuana) has taken place worldwide for centuries. In Canada, legalization of cannabis in October 2018 for the medicinal and recreational markets has spurned interest in large-scale growing. This increased production has seen a rise in the incidence and severity of plant pathogens, causing a range of previously unreported diseases. The objective of this review is to highlight the important diseases currently affecting the cannabis and hemp industries in North America and to discuss various mitigation strategies. Progress in molecular diagnostics for pathogen identification and determining inoculum sources and methods of pathogen spread have provided useful insights. Sustainable disease management approaches include establishing clean planting stock, modifying environmental conditions to reduce pathogen development, implementing sanitation measures, and applying fungal and bacterial biological control agents. Fungicides are not currently registered for use and hence there are no published data on their efficacy. The greatest challenge remains in reducing microbial loads (colony-forming units) on harvested inflorescences (buds). Contaminating microbes may be introduced during the cultivation and postharvest phases, or constitute resident endophytes. Failure to achieve a minimum threshold of microbes deemed to be safe for utilization of cannabis products can arise from conventional and organic cultivation methods, or following applications of beneficial biocontrol agents. The current regulatory process for approval of cannabis products presents a challenge to producers utilizing biological control agents for disease management. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/ps.6307

Emerging Diseases of Cannabis sativa and Sustainable Management.

For nearly a century, Cannabis has been stigmatized and criminalized across the globe, but in recent years, there has been a growing interest in Cannabis due to the therapeutic potential of phytocannabinoids. With this emerging interest in Cannabis, concerns have arisen about the possible contaminations of hemp with pesticides, heavy metals, microbial pathogens, and carcinogenic compounds during the cultivation, manufacturing, and packaging processes. This is of particular concern for those turning to Cannabis for medicinal purposes, especially those with compromised immune systems. This review aims to provide types of contaminants and examples of Cannabis contamination using case studies that elucidate the medical consequences consumers risk when using adulterated Cannabis products. Thus, it is imperative to develop universal standards for cultivation and testing of products to protect those who consume Cannabis.

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Cannabis Contaminants Limit Pharmacological Use of Cannabidiol.

Cannabis legalization has occurred in several countries worldwide. Along with steadily growing research in Cannabis healthcare science, there is an increasing interest for scientific-based knowledge in plant microbiology and food science, with work connecting the plant microbiome and plant health to product quality across the value chain of cannabis. This review paper provides an overview of the state of knowledge and challenges in Cannabis science, and thereby identifies critical risk management and safety issues in order to capitalize on innovations while ensuring product quality control. It highlights scientific gap areas to steer future research, with an emphasis on plant-microbiome sciences committed to using cutting-edge technologies for more efficient Cannabis production and high-quality products intended for recreational, pharmaceutical, and medicinal use.

Scientific Prospects for Cannabis-Microbiome Research to Ensure Quality and Safety of Products.

Cannabis sativa L (hemp, marijuana) produces male and female inflorescences on different plants (dioecious) and therefore the plants are obligatory out-crossers In commercial production, marijuana plants are all genetically female; male plants are destroyed as seed formation reduces flower quality Spontaneously occurring hermaphroditic inflorescences, in which pistillate flowers are accompanied by formation of anthers, leads to undesired seed formation; the mechanism for this is poorly understood We studied hermaphroditism in several marijuana strains with three objectives: (i) to compare the morphological features of this unique phenotype with normal male flowers; (ii) to assess pollen and seed viability from hermaphroditic flowers; and (iii) to assess the effect of hermaphroditism on progeny male:female (sex) ratios and on genetic variation using molecular methods The morphological features of anthers, pollen production and germination in hermaphroditic flowers and in staminate inflorescences on male plants were compared using light and scanning electron microscopy Seeds produced on hermaphroditic plants and seeds derived from cross-fertilization were germinated and seedlings were compared for gender ratios using a PCR-based assay as well as for the extent of genetic variation using six ISSR primers Nei's index of gene diversity and Shannon's Information index were compared for these two populations The morphology of anthers and pollen formation in hermaphroditic inflorescences was similar to that in staminate flowers Seedlings from hermaphroditic seeds, and anther tissues, showed a female genetic composition while seedlings derived from cross-fertilized seeds showed a 1:1 male:female sex expression ratio Uniquely, hermaphroditic inflorescences produced seeds which gave rise only to genetically female plants In PCR assays, a 540 bp size fragment was present in male and female plants, while a 390 bp band was uniquely associated with male plants Sequence analysis of these fragments revealed the presence of Copia-like retrotransposons within the C sativa genome which may be associated with the expression of male or female phenotype In ISSR analysis, the percentage of polymorphic loci ranged from 44 to 72% in hermaphroditic and cross-fertilized populations Nei's index of gene diversity and Shannon's Information index were not statistically different for both populations The extent of genetic variation after one generation of selfing in the progeny from hermaphroditic seed is similar to that in progeny from cross-fertilized seeds

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Hermaphroditism in Marijuana (Cannabis sativa L.) Inflorescences - Impact on Floral Morphology, Seed Formation, Progeny Sex Ratios, and Genetic Variation.

Plant pathogens infecting marijuana (Cannabis sativa L.) plants reduce growth of the crop by affecting the roots, crown and foliage. In addition, fungi (molds) that colonize the inflorescences (buds) during development or after harvest, and which colonize internal tissues as endophytes, can reduce product quality. The pathogens and molds that affect C. sativa grown hydroponically indoors (in environmentally controlled growth rooms and greenhouses) and field-grown plants were studied over multiple years of sampling. A PCR-based assay using primers for the internal transcribed spacer region (ITS) of ribosomal DNA confirmed identity of the cultures. Root-infecting pathogens included Fusarium oxysporum, F. solani, F. brachygibbosum, Pythium dissotocum, P. myriotylum and P. aphanidermatum, which caused root browning, discoloration of the crown and pith tissues, stunting and yellowing of plants, and in some instances, plant death. On the foliage, powdery mildew, caused by Golovinomyces cichoracearum, was the major pathogen observed. On inflorescences, penicillium bud rot (caused by P. olsonii and P. copticola), botrytis bud rot (B. cinerea) and fusarium bud rot (F. solani, F. oxysporum) were present to varying extents. Endophytic fungi present in crown, stem and petiole tissues included soil-colonizing and cellulolytic fungi, such as species of Chaetomium, Trametes, Trichoderma, Penicillium and Fusarium. Analysis of air samples in indoor growing environments revealed that species of Penicillium, Cladosporium, Aspergillus, Fusarium, Beauvaria, and Trichoderma were present. The latter two species were the result of the application of biocontrol products for control of insects and diseases, respectively. Fungal communities present in unpasteurized coconut fibre (coco) growing medium are potential sources of mold contamination on cannabis plants. Swabs taken from greenhouse-grown and indoor buds pre- and post-harvest revealed the presence of Cladosporium and up to five species of Penicillium, as well as low levels of Alternaria species. Mechanical trimming of buds caused an increase in the frequency of Penicilllium species, presumably by providing entry points through wounds or spreading endophytes from pith tissues. Aerial distribution of pathogen inoculum and mold spores and dissemination through vegetative propagation, are important methods of spread, and entry through wound sites on roots, stems and bud tissues facilitates pathogen establishment on cannabis plants.

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Pathogens and Molds Affecting Production and Quality of Cannabis sativa L.

Tissue culture approaches are widely used in crop plants for the purposes of micropropagation, regeneration of plants through organogenesis, obtaining pathogen-free plantlets from meristem culture, and developing genetically modified plants. In this research, we evaluated variables that can influence the success of shoot growth and plantlet production in tissue cultures of drug-type Cannabis sativa L. (marijuana). Various sterilization methods were tested to ensure shoot development from nodal explants by limiting the frequency of contaminating endophytes, which otherwise caused the death of explants. Seven commercially grown tetrahydrocannabinol (THC)-containing cannabis genotypes (strains) showed significant differences in response to shoot growth from meristems and nodal explants on Murashige and Skoog (MS) medium containing thidiazuron (1 μM) and naphthaleneacetic acid (0.5 μM) plus 1% activated charcoal. The effect of Driver and Kuniyuki Walnut (DKW) or MS basal salts in media on shoot length and leaf numbers from nodal explants was compared and showed genotype dependency with regard to the growth response. To obtain rooted plantlets, shoots from meristems and nodal explants of genotype Moby Dick were evaluated for rooting, following the addition of sodium metasilicate, silver nitrate, indole-3-butyric acid (IBA), kinetin, or 2,4-D. Sodium metasilicate improved the visual appearance of the foliage and improved the rate of rooting. Silver nitrate also promoted rooting. Following acclimatization, plantlet survival in hydroponic culture, peat plugs, and rockwool substrate was 57, 76, and 83%, respectively. The development of plantlets from meristems is described for the first time in C. sativa and has potential for obtaining pathogen-free plants. The callogenesis response of leaf explants of 11 genotypes on MS medium without activated charcoal was 35% to 100%, depending on the genotype; organogenesis was not observed. The success in recovery of plantlets from meristems and nodal explants is influenced by cannabis genotype, degree of endophytic contamination of the explants, and frequency of rooting. The procedures described here have potential applications for research and commercial utility to obtain plantlets in stage 1 tissue cultures of C. sativa.

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Variables Affecting Shoot Growth and Plantlet Recovery in Tissue Cultures of Drug-Type Cannabis sativa L.

Fusarium graminearum, a filamentous fungus, and causal agent of Fusarium head blight (FHB) in wheat and other cereals, leads to significant economic losses globally. This study aimed to investigate the roles of specific genes in F. graminearum virulence using CRISPR/Cas9-mediated gene deletions. Illumina sequencing was used to characterize the genomic changes due to editing. Unexpectedly, a large-scale deletion of 525,223 base pairs on chromosome 2, comprising over 222 genes, occurred in two isolates. Many of the deleted genes were predicted to be involved in essential molecular functions, such as oxidoreductase activity, transmembrane transporter activity, hydrolase activity, as well as biological processes, such as carbohydrate metabolism and transmembrane transport. Despite the substantial loss of genetic material, the mutant isolate exhibited normal growth rates and virulence on wheat under most conditions. However, growth rates were significantly reduced under high temperatures and on some media. Additionally, wheat inoculation assays using clip dipping, seed inoculation, and head point inoculation methods were performed. No significant differences in virulence were observed, suggesting that these genes were not involved in infection or alternative compensatory pathways, and allow the fungi to maintain pathogenicity despite the extensive genomic deletion.

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Unanticipated Large-Scale Deletion in Fusarium graminearum Genome Using CRISPR/Cas9 and Its Impact on Growth and Virulence

We established transformation technologies using Agrobacterium tumefaciens to insert foreign genes into high THC-containing cannabis (Cannabis sativa L.). The Arabidopsis non-expressor of pathogenesis-related protein 1 (AtNPR1) gene was selected as a potentially useful agronomic gene which was linked to the bar gene from Streptomyces that encodes herbicide resistance. We investigated how Agrobacterium strains (EHA105 and GV3101), glufosinate concentrations, explant source, and light intensity, affected transformation frequency (TF). Transformation was confirmed by RT-PCR with primers for the NPR1 or bar genes. Glufosinate at 0.5-1 mg/L inhibited growth of non-transformed calli within 8 weeks following A. tumefaciens infection. Strain EHA105 yielded a higher TF compared to strain GV3101. Whole leaflets yielded a higher TF compared to sectioned leaf explants with strain GV3101. However, this effect was not seen with EHA105. Petiole segments showed a higher TF than leaf sections with strain EHA105. Placing explants under light or dark conditions did not affect TF, which ranged from 5 % to 95 % in different experiments. This is the first report of successful transformation of two high THC-containing C. sativa genotypes with two foreign genes simultaneously - AtNPR1 and bar. The recovery of plantlets from transgenic calli was not attempted and awaits further research.

Agrobacterium-mediated transformation of THC-containing Cannabis sativa L. yields a high frequency of transgenic calli expressing bialaphos resistance and non-expressor of PR1 (NPR1) genes

Janesse E. Holmes

Papers

Pathogens and Molds Affecting Production and Quality of Cannabis sativa L.

Hermaphroditism in Marijuana (Cannabis sativa L.) Inflorescences - Impact on Floral Morphology, Seed Formation, Progeny Sex Ratios, and Genetic Variation.

Variables Affecting Shoot Growth and Plantlet Recovery in Tissue Cultures of Drug-Type Cannabis sativa L.

Unanticipated Large-Scale Deletion in Fusarium graminearum Genome Using CRISPR/Cas9 and Its Impact on Growth and Virulence

Agrobacterium-mediated transformation of THC-containing Cannabis sativa L. yields a high frequency of transgenic calli expressing bialaphos resistance and non-expressor of PR1 (NPR1) genes