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

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

Total yeast and mold levels in high THC-containing cannabis (Cannabis sativa L.) inflorescences are influenced by genotype, environment, and pre-and post-harvest handling practices

Total yeast and mold (TYM) levels in inflorescences of high THC-containing Cannabis sativa (cannabis) are regulated to ensure that medicinal and recreational users, especially those with immunocompromised systems, are not exposed to potentially harmful levels. In North America, the limits imposed range from 1,000–10,000 cfu/g of dried product to 50,000–100,000 cfu/g, depending on the jurisdiction. Factors affecting a build-up of TYM in cannabis inflorescences have not been previously researched. In this study, >2,000 fresh and dried samples were assayed for TYM over a 3-year period (2019–2022) to identify specific factors which can contribute to TYM levels. Greenhouse-grown inflorescences were sampled before and after commercial harvest, homogenized for 30 s, and plated onto potato dextrose agar (PDA) with 140 mg/L streptomycin sulfate. Colony-forming-units (cfu) were rated after 5 days of incubation at 23°C under 10–14 h light. PDA provided more consistent counts of cfu compared to Sabouraud dextrose and tryptic soy agars. The predominant fungal genera identified by PCR of the ITS1-5.8S-ITS2 region of rDNA were Penicillium, Aspergillus, Cladosporium, and Fusarium. In addition, four yeast genera were recovered. In total, 21 species of fungi and yeasts constituted the total cfu present in the inflorescences. The variables that significantly (p < 0.05) increased these TYM levels in inflorescences were: the genotype (strain) grown, presence of leaf litter in the greenhouse, harvesting activity by workers, genotypes with a higher abundance of stigmatic tissues and inflorescence leaves, higher temperature and relative humidity within the inflorescence microclimate, time of year (May–October), method of drying buds after harvest, and inadequate drying of buds. The variables which significantly (p < 0.05) decreased TYM in samples were: genotypes with lower numbers of inflorescence leaves, air circulation achieved by fans during inflorescence maturation, harvesting during November–April, hang-drying of entire inflorescence stems, and drying to a moisture content of 12–14% (water activity of 0.65–0.7) or lower which was inversely correlated with cfu levels. Under these conditions, the majority of dried commercial cannabis samples contained <1,000–5,000 cfu/g. Our findings indicate that TYM in cannabis inflorescences are the result of a dynamic interaction between genotype, environment, and post-harvest handling methods. Some of these factors may be altered by cannabis producers to reduce the potential build-up of these microbes. Among the 21 fungal and yeast species recovered from greenhouse-grown cannabis inflorescences, a few could pose a potential threat to human health, while many do not and they could provide beneficial interactions within the cannabis plant. The currently recommended plating methods onto agar media and enumeration of total cfu are unable to distinguish between these two groups

Variables Affecting Shoot Growth and Plantlet Recovery in Tissue Cultures of Drug-Type 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

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

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, Fusarium solani, Fusarium brachygibbosum, Pythium dissotocum, Pythium myriotylum, and Pythium 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 Penicillium olsonii and Penicillium copticola), Botrytis bud rot (Botrytis 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, Beauveria, 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 (coco) fiber 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 Penicillium 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

Genetic engineering of plants to enhance resistance to fungal pathogens—a review of progress and future prospects

Abstract: Recent applications of techniques in plant molecular biology and biotechnology to the study of hostpathogen interactions have resulted in the identification and cloning of numerous genes involved in the defense responses of plants following pathogen infection. These include: genes that express proteins, peptides, or antimicrobial compounds that are directly toxic to pathogens or that reduce their growth in situ; gene products that directly inhibit pathogen virulence products or enhance plant structural defense genes, that directly or indirectly activate general plant defense responses; and resistance genes involved in the hypersensitive response and in the interactions with avirulence factors. The introduction and expression of these genes, as well as of antimicrobial genes from nonplant sources, in a range of transgenic plant species have shown that the development of fungal pathogens can be significantly reduced. The extent of disease reduction varies with the strategy employed as well as with the characteristics of the fungal pathogen, and disease control has never been complete. Manipulation of salicylic acid, ethylene, and cytokinin levels in transgenic plants have provided some interesting results with regard to enhanced disease tolerance or susceptibility. The complex interactions among the expressed gene product, plant species, and fungal pathogen indicate that the response of transgenic plants cannot be readily predicted. Combinations of defense gene products have shown considerably more promise in reducing disease than single-transgene introductions. The use of tissue-specific or pathogen-inducible promoters, and the engineered expression of resistance genes, synthetic antimicrobial peptides, and elicitor molecules that induce defense responses have the potential to provide commercially useful broad-spectrum disease resistance in the not-too-distant future. The issues and challenges that will need to be addressed prior to the widespread utilization of these transgenic plants are highlighted. Key words: antifungal proteins, antimicrobial peptides, biotechnology, elicitors, hypersensitive response, pathogenesis-related proteins, phytoalexins, resistance genes, transgenic plants. 235 Résumé : Les application récentes au domaine végétal des techniques de la biologie moléculaire et de la biotechnologie pour l'étude des interactions hôtes-pathogènes ont permis d'identifier et de cloner plusieurs gènes impliqués dans les réponses de défense des plantes par suite d'une infection par un agent pathogène. Ceux-ci comprennent : des gènes qui codent pour des protéines, des peptides ou des composés antimicrobiens qui ont une toxicité directe ou réduisent la croissance in situ des agents pathogènes; des produits géniques qui inhibent directement des produits de virulence des agents pathogènes ou stimulent les gènes de défense structurale des plantes, qui activent directement ou indirectement les réponses générales de défense des plantes et des gènes de résistance impliqués dans la réaction d'hypersensibilité et dans les interactions avec les facteurs d'avirulence. L'introduction et l'expression de ces gènes, ainsi que celles de gènes antimicrobiens de source non végétale, dans une gamme d'espèces végétales transgéniques ont montré que le développement des champignons pathogènes peut être significativement réduit. L'ampleur de la réduction de la maladie dépends de la stratégie utilisée aussi bien que des caractéristiques du champignon pathogène; une répression complète de la maladie n'a pas encore été atteinte. La manipulation des niveaux d'acide salicylique, d'éthylène et de cytokinines dans des plantes transgéniques a fourni quelques résultats intéressants concernant la tolérance ou la sensibilité accrues à la maladie. Les interactions complexes entre les produits géniques exprimés, les espèces végétales et les champignons pathogènes laissent voir que la réponse des plantes transgéniques ne peut être parfaitement prédite. Les combinaisons de produits géniques de défense ont été beaucoup plus prometteuses que les introductions d'un seul transgène pour lutter contre les maladies. L'emploi de promoteurs spécifiques à un tissu ou inductibles par les agents pathogènes et le génie appliqué à l'expression de gènes de résistance, de peptides synthétiques antimicrobiens et de molécules inductrices qui stimulent les réponses de défense ont le potentiel pour fournir commercialement, dans un futur pas si lointain, un moyen pratique pour résister à un large spectre de maladies. Les problèmes et les défis qui devront être surmontés avant d'arriver à une utilisation à grande échelle de ces plantes transgéniques sont mis en évidence

Hemp (Cannabis sativa L.)

Hemp (Cannabis sativa L.) suspension culture cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective agent, mannose, whereas cells not expressing the gene are incapable of using the carbon source and will stop growing. Callus masses proliferating on selection were screened for PMI expression using a chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines and the presence of the PMI gene was confirmed using polymerase chain reaction and Southern hybridization. Using this method, an average transformation frequency of 31.23% ± 0.14 was obtained for all transformation experiments, with a range of 15.1 to 55.3%

Integrated Management of Pathogens and Microbes in <i>Cannabis sativa</i> L. (Cannabis) under Greenhouse Conditions

The increased cultivation of high THC-containing Cannabis sativa L. (cannabis), particularly in greenhouses, has resulted in a greater incidence of diseases and molds that can negatively affect the growth and quality of the crop. Among them, the most important diseases are root rots (Fusarium and Pythium spp.), bud rot (Botrytis cinerea), powdery mildew (Golovinomyces ambrosiae), cannabis stunt disease (caused by hop latent viroid), and a range of microbes that reduce post-harvest quality. An integrated management approach to reduce the impact of these diseases/microbes requires combining different approaches that target the reproduction, spread, and survival of the associated pathogens, many of which can occur on the same plant simultaneously. These approaches will be discussed in the context of developing an integrated plan to manage the important pathogens of greenhouse-grown cannabis at different stages of plant development. These stages include the maintenance of stock plants, propagation through cuttings, vegetative growth of plants, and flowering. The cultivation of cannabis genotypes with tolerance or resistance to various pathogens is a very important approach, as well as the maintenance of pathogen-free stock plants. When combined with cultural approaches (sanitation, management of irrigation, and monitoring for diseases) and environmental approaches (greenhouse climate modification), a significant reduction in pathogen development and spread can be achieved. The use of preventive applications of microbial biological control agents and reduced-risk biorational products can also reduce disease development at all stages of production in jurisdictions where they are registered for use. The combined use of promising strategies for integrated disease management in cannabis plants during greenhouse production will be reviewed. Future areas for research are identified

Hemp (Cannabis sativa L.)

Hemp (Cannabis sativa L.) suspension culture cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective agent mannose, whereas cells not expressing the gene are incapable of using the carbon source and will stop growing. Callus masses proliferating on selection medium were screened for PMI expression using a chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines, and the presence of the PMI gene was confirmed using PCR and Southern hybridization. Using this method, an average transformation frequency of 31.23 % ± 0.14 was obtained for all transformation experiments, with a range of 15.1–55.3 %