Comparative proteomic and metabolomic analyses reveal stress responses of hemp to salinity.

KEY MESSAGE: Integrated omics analyses outline the cellular and metabolic events of hemp plants in response to salt stress and highlight several photosynthesis and energy metabolism related pathways as key regulatory points. Soil salinity affects many physiological processes of plants and leads to crop yield losses worldwide. For hemp, a crop that is valued for multiple aspects, such as its medical compounds, fibre, and seed, a comprehensive understanding of its salt stress responses is a prerequisite for resistance breeding and tailoring its agronomic performance to suit certain industrial applications. Here, we first observed the phenotype of salt-stressed hemp plants and found that under NaCl treatment, hemp plants displayed pronounced growth defects, as indicated by the significantly reduced average height, number of leaves, and chlorophyll content. Next, we conducted comparative proteomics and metabolomics to dissect the complex salt-stress response mechanisms. A total of 314 proteins and 649 metabolites were identified to be differentially behaving upon NaCl treatment. Functional classification and enrichment analysis unravelled that many differential proteins were proteases associated with photosynthesis. Through metabolic pathway enrichment, several energy-related pathways were found to be altered, such as the biosynthesis and degradation of branched-chain amino acids, and our network analysis showed that many ribosomal proteins were involved in these metabolic adaptations. Taken together, for hemp plants, influences on chloroplast function probably represent a major toxic effect of salinity, and modulating several energy-producing pathways possibly through translational regulation is presumably a key protective mechanism against the negative impacts. Our data and analyses provide insights into our understanding of hemp's stress biology and may lay a foundation for future functional genomics studies.

A FLOWERING LOCUS T ortholog is associated with photoperiod-insensitive flowering in hemp (Cannabis sativa L.).

Hemp (Cannabis sativa L.) is an extraordinarily versatile crop, with applications ranging from medicinal compounds to seed oil and fibre products. Cannabis sativa is a short-day plant, and its flowering is highly controlled by photoperiod. However, substantial genetic variation exists for photoperiod sensitivity in C. sativa, and photoperiod-insensitive ("autoflower") cultivars are available. Using a bi-parental mapping population and bulked segregant analysis, we identified Autoflower2, a 0.5 Mbp locus significantly associated with photoperiod-insensitive flowering in hemp. Autoflower2 contains an ortholog of the central flowering time regulator FLOWERING LOCUS T (FT) from Arabidopsis thaliana which we termed CsFT1. We identified extensive sequence divergence between alleles of CsFT1 from photoperiod-sensitive and insensitive cultivars of C. sativa, including a duplication of CsFT1 and sequence differences, especially in introns. Furthermore, we observed higher expression of one of the CsFT1 copies found in the photoperiod-insensitive cultivar. Genotyping of several mapping populations and a diversity panel confirmed a correlation between CsFT1 alleles and photoperiod response, affirming that at least two independent loci involved in the photoperiodic control of flowering, Autoflower1 and Autoflower2, exist in the C. sativa gene pool. This study reveals the multiple independent origins of photoperiod insensitivity in C. sativa, supporting the likelihood of a complex domestication history in this species. By integrating the genetic relaxation of photoperiod sensitivity into novel C. sativa cultivars, expansion to higher latitudes will be permitted, thus allowing the full potential of this versatile crop to be reached.

Physiological and cannabinoid responses of hemp (Cannabis sativa) to rock phosphate dust under tropical conditions.

Growing a high-value crop such as industrial hemp (Cannabis sativa L.) in post-mining environments is economically and environmentally attractive but faces a range of biotic and abiotic challenges. An opportunity to investigate the cultivation of C. sativa presented itself as part of post-mining activities on Christmas Island (Australia) to profitably utilise disused phosphate (PS) quarries. Challenges to plant growth and cadmium (Cd) uptake were addressed in this study using potted plants under fully controlled conditions in a growth chamber. A complete nutritional spectrum, slow-release fertiliser was applied to all plants as a control treatment, and two levels of rock PS dust, a waste product of PS mining that contains 35% phosphorus (P) and 40ppm of naturally occurring Cd, were applied at 54 and 162gL-1 . After 12weeks, control plants (no PS dust) significantly differed in phenological development, with no flower production, lower aboveground biomass and reduced photosynthesis efficiency than those with P applied as rock dust. Compared with the controls, the 54gL-1 level of P dust increased shoot biomass by 38%, while 162gL-1 increased shoot biomass by 85%. The concentration of Δ9 -tetrahydrocannabinol also increased with the higher P levels. Cd uptake from PS dust by C. sativa was substantial and warrants further investigation. However, there was no increase in Cd content between the 54 and 162gL-1 application rates in seed and leaf. Results indicate that hemp could become a high-value crop on Christmas Island, with the readily available rock PS dust providing a source of P.

Spiritual Benefit from Cannabis.

Like many mind-altering plants, cannabis has been part of spiritual practices for thousands of years. It has deep roots in Hinduism, Islam, Rastafarianism, and indigenous traditions in Asia, Africa, and elsewhere. Yet almost no attention has been given to how contemporary adults employ it spiritually. A sample of 1087 participants (mean age = 38.9) completed an online survey assessing their use of cannabis and other substances, as well as spiritual and psychological characteristics. Spiritual benefit from cannabis was reported by 66.1% of the sample, and 5.5% reported it had sometimes been a spiritual hindrance. A MANOVA showed that those who reported spiritual benefit differed significantly from those who did not on several outcome variables, and a post hoc descriptive discriminant analysis revealed that expansiveness motivation, non-theistic daily spiritual experience, meditation frequency, and two mindfulness facets contributed most to differentiating the two groups. The majority of the sample (63%) was free of cannabis use disorder. Compared to disordered groups, the non-disordered group was significantly older and scored lower on experiential avoidance, psychological distress, and several motives for use. Results suggest that spiritual motives for cannabis use may be widespread. Implications for future research on spiritual use of cannabis are discussed.

Dioecious hemp (Cannabis sativa L.) plants do not express significant sexually dimorphic morphology in the seedling stage.

Some economically important crop species are dioecious, producing pollen and ovules on distinct, unisexual, individuals. On-the-spot diagnosis of sex is important to breeders and farmers for crop improvement and maximizing yield, yet diagnostic tools at the seedling stage are understudied and lack a scientific basis. Understanding sexual dimorphism in juvenile plants may provide key ecological, evolutionary and economic insights into dioecious plant species in addition to improving the process of crop cultivation. To address this gap in the literature, we asked: can we reliably differentiate males, females, and co-sexual individuals based on seedling morphology in Cannabis sativa, and do the traits used to distinguish sex at this stage vary between genotypes? To answer these questions, we collected data on phenotypic traits of 112 C. sativa plants (50 female, 52 male, 10 co-sexuals) from two hemp cultivars (CFX-1, CFX-2) during the second week of vegetative growth and used ANOVAs to compare morphology among sexes. We found males grew significantly longer hypocotyls than females by week 2, but this difference depended on the cultivar investigated. Preliminary evidence suggests that co-sexual plants may be distinguished from male and female plants using short hypocotyl length and seedling height, although this relationship requires more study since sample sizes of co-sexual plants were small. In one of the cultivars, two-week old male plants tend to produce longer hypocotyls than other plants, which may help to identify these plants prior to anthesis. We call for increased research effort on co-sexual plants, given their heavy economic cost in industrial contexts and rare mention in the literature. Our preliminary data suggests that short hypocotyl length may be an indicator of co-sexuality. These results are the first steps towards developing diagnostic tools for predicting sex using vegetative morphology in dioecious species and understanding how sexual dimorphism influences phenotype preceding sexual maturity.

Characteristics of the Diploid, Triploid, and Tetraploid Versions of a Cannabigerol-Dominant F1 Hybrid Industrial Hemp Cultivar, Cannabis sativa 'Stem Cell CBG'.

Hemp (Cannabis sativa L.) has recently become an important crop due to the growing market demands for products containing cannabinoids. Unintended cross-pollination of C. sativa crops is one of the most important threats to cannabinoid production and has been shown to reduce cannabinoid yield. Ploidy manipulation has been used in other crops to improve agronomic traits and reduce fertility; however, little is known about the performance of C. sativa polyploids. In this study, colchicine was applied to two proprietary, inbred diploid C. sativa inbred lines, 'TS1-3' and 'P163', to produce the tetraploids 'TS1-3 (4x)' and 'P163 (4x)'. The diploid, triploid, and tetraploid F1 hybrids from 'TS1-3' × 'P163', 'TS1-3 (4x)' × 'P163', and 'TS1-3 (4x)' × 'P163 (4x)' were produced to test their fertilities, crossing compatibilities, and yields. The results indicated a reduction in fertility in the triploids and the tetraploids, relative to their diploid counterparts. When triploids were used as females, seed yields were less than 2% compared to when diploids were used as females; thus, triploids were determined to be female infertile. The triploids resulting from the crosses made herein displayed increases in biomass and inflorescence weight compared to the diploids created from the same parents in a field setting. Statistical increases in cannabinoid concentrations were not observed. Lastly, asymmetric crossing compatibility was observed between the diploids and the tetraploids of the genotypes tested. The results demonstrate the potential benefits of triploid C. sativa cultivars in commercial agriculture.

Physiological and transcriptome analyses for assessing the effects of exogenous uniconazole on drought tolerance in hemp (Cannabis sativa L.).

Uniconazole (S-(+)-uniconazole), a plant growth retardant, exerts key roles in modulating growth and development and increasing abiotic stress tolerance in plants. However, the underlying mechanisms by which uniconazole regulates drought response remain largely unknown. Here, the effects of exogenous uniconazole on drought tolerance in hemp were studied via physiological and transcriptome analyses of the drought-sensitive industrial hemp cultivar Hanma No. 2 grown under drought stress. Exogenous uniconazole treatment increased hemp tolerance to drought-induced damage by enhancing chlorophyll content and photosynthesis capacity, regulating activities of enzymes involved in carbon and nitrogen metabolism, and altering endogenous hormone levels. Expression of genes associated with porphyrin and chlorophyll metabolism, photosynthesis-antenna proteins, photosynthesis, starch and sucrose metabolism, nitrogen metabolism, and plant hormone signal transduction were significantly regulated by uniconazole compared with that by control (distilled water) under drought stress. Numerous genes were differentially expressed to increase chlorophyll content, enhance photosynthesis, regulate carbon-nitrogen metabolism-related enzyme activities, and alter endogenous hormone levels. Thus, uniconazole regulated physiological and molecular characteristics of photosynthesis, carbon-nitrogen metabolism, and plant hormone signal transduction to enhance drought resistance in industrial hemp.

The characterization of key physiological traits of medicinal cannabis (Cannabis sativa L.) as a tool for precision breeding.

BACKGROUND: For millennia, drug-type cannabis strains were extensively used for various medicinal, ritual, and inebriant applications. However, cannabis prohibition during the last century led to cultivation and breeding activities being conducted under clandestine conditions, while scientific development of the crop ceased. Recently, the potential of medicinal cannabis has been reacknowledged and the now expanding industry requires optimal and scientifically characterized varieties. However, scientific knowledge that can propel this advancement is sorely lacking. To address this issue, the current study aims to provide a better understanding of key physiological and phenological traits that can facilitate the breeding of advanced cultivars. RESULTS: A diverse population of 121 genotypes of high-THC or balanced THC-CBD ratio was cultivated under a controlled environment facility and 13 plant parameters were measured. No physiological association across genotypes attributed to the same vernacular classification was observed. Floral bud dry weight was found to be positively associated with plant height and stem diameter but not with days to maturation. Furthermore, the heritability of both plant height and days to maturation was relatively high, but for plant height it decreased during the vegetative growth phase. To advance breeding efficacy, a prediction equation for forecasting floral bud dry weight was generated, driven by parameters that can be detected during the vegetative growth phase solely. CONCLUSIONS: Our findings suggest that selection for taller and fast-growing genotypes is likely to lead to an increase in floral bud productivity. It was also found that the final plant height and stem diameter are determined by 5 independent factors that can be used to maximize productivity through cultivation adjustments. The proposed prediction equation can facilitate the selection of prolific genotypes without the completion of a full cultivation cycle. Future studies that will associate genome-wide variation with plants morphological traits and cannabinoid profile will enable precise and accelerated breeding through genomic selection approaches.

Biotechnological Approaches on Two High CBD and CBG Cannabis sativa L. (Cannabaceae) Varieties: In Vitro Regeneration and Phytochemical Consistency Evaluation of Micropropagated Plants Using Quantitative 1H-NMR.

High cannabidiol (CBD) and cannabigerol (CBG) varieties of Cannabis sativa L., a species with medicinal properties, were regenerated in vitro. Explants of nodal segments including healthy axillary bud, after sterilization, were placed in Murashige-Skoog (MS) culture medium. The shoots formed after 30 days were subcultured in full- or half-strength MS medium supplemented with several concentrations of 6-benzyl-amino-purine (BA) or thidiazuron (TDZ). The highest average number and length of shoots was achieved when both full and half-strength MS media were supplemented with 4.0 µM BA. The presence of 4.0 µM TDZ showed also comparable results. BA and TDZ at concentrations of 4.0, 8.0 µM and 2.0, 4.0 µM respectively, displayed the maximum shooting frequency. The new shoots were transferred on the same media and were either self-rooted or after being enhanced with different concentrations of indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Presence of 2.0 or 4.0 µM IBA or 4.0 µM NAA resulted to the optimum rooting rates. The maximum average number and length of roots per shoot was observed when the culture media was supplemented with 4.0 µM IBA or NAA. Approximately 92% of the plantlets were successfully established and acclimatized in field. The consistency of the chemical profile of the acclimatized in vitro propagated clones was assessed using quantitative 1H-NMR high throughput screening. In each variety, analysis of the micropropagated plant in comparison with the mother plant showed no statistically significant differences (p ≤ 0.05) in CBD+ cannabidiolic acid (CBDA) and CBG+ cannabigerolic acid (CBGA) content respectively, thus indicating stability of their chemical profile.

[Cannabis: Similarities and differences with tobacco]. / Le cannabis : similitudes et différences avec le tabac.

Nicotine is the specific psychoactive substance of tobacco while tetrahydrocannabinol (THC) is the specific component of cannabis. The inhalation technique of cannabis is different from that of tobacco smoking: the volume of puffs is larger, inhalation is deeper, and pulmonary retention time is longer. Cannabis addiction is difficult to evaluate, both products often being smoked concomitantly. The principle physical side effects of cannabis affect organs and functions in a similar way to tobacco: pulmonary, cardiovascular, endocrine and stomatological. Gastrointestinal complications such as cannabinoid hyperemesis are specific to cannabis. Some psychological effects of THC may be acute (altered time and space perception, sensory disability, decreased vigilance, mood and dissociative disorders, hallucinations and delirium, impaired learning and memory, impaired cognitive and motor performance, panic attacks and anxiety) or chronic (lack of motivation, disorganisation of thoughts, increase in frequency and severity of schizophrenic crises). Cannabis can also be implicated in traffic and workplace accidents. Synthetic cannabinoids have increased psychotropic and somatic effects due to a greater affinity for brain cannabinoid receptors.

Photochemical efficiency is negatively correlated with the Δ9- tetrahydrocannabinol content in Cannabis sativa L.

Cannabis sativa L is an important plant, which is a source of durable fibers, nutritious seeds, and medicinally important phytocannabinoids including Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Light has shown to be a key modulator of biomass and cannabinoid yield suggesting responsive photochemical machinery. The present study was envisaged to understand the effect of the increasing levels of metabolic THC on the photochemical efficiency in Cannabis. The chlorophyll a fluorescence kinetics, photosynthetic pigments and immuno-detection of the photosynthetic machinery was analyzed on seven accessions from different environments, in conjunction with the cannabinoid content. All the accessions were clearly divided into three groups based on their relative content of CBD and THC. Group I with (CBD/THC > 1) had a clear advantage in terms of the damage to the D1, RbCL and Lhc1 protein holo-complex. Performance indicators of photochemistry based on the OJIP kinetics suggested a stoichiometrically negative correlation with the THC content. Zeaxanthin-dependent quenching is primarily responsible for lower NPQ in Group III with high THC content (THC > 6%). The THC treatment on Arabidopsis thaliana also suggested dose-dependent decrease in the photochemical efficiency suggesting the exclusivity of THC in causing the response. This resulted in the damage of photosynthetic machinery and the generation of free radicals, thereby compromising the yield. The study also opens a new screening method for Cannabis, based on cannabinoid content.

Phytocannabinoids: Useful Drugs for the Treatment of Obesity? Special Focus on Cannabidiol.

Currently, an increasing number of diseases related to insulin resistance and obesity is an alarming problem worldwide. It is well-known that the above states can lead to the development of type 2 diabetes, hypertension, and cardiovascular diseases. An excessive amount of triacylglycerols (TAGs) in a diet also evokes adipocyte hyperplasia and subsequent accumulation of lipids in peripheral organs (liver, cardiac muscle). Therefore, new therapeutic methods are constantly sought for the prevention, treatment and alleviation of symptoms of the above mentioned diseases. Currently, much attention is paid to Cannabis derivatives-phytocannabinoids, which interact with the endocannabinoid system (ECS) constituents. Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are the most abundant compounds of Cannabis plants and their therapeutic application has been suggested. CBD is considered as a potential therapeutic agent due to its anti-inflammatory, anti-oxidant, anti-tumor, neuroprotective, and potential anti-obesity properties. Therefore, in this review, we especially highlight pharmacological properties of CBD as well as its impact on obesity in different tissues.

Cannabis sativa.

Schilling et al. introduce and discuss Cannabis.

Has the "M" word been framed? Marijuana, cannabis, and public opinion.

Over the past two decades, a growing cadre of US states has legalized the drug commonly known as "marijuana." But even as more states legalize the drug, proponents of reform have begun to shun the term "marijuana" in favor of the term "cannabis." Arguing that the "M" word has been tainted and may thus dampen public support for legalization, policy advocates have championed "cannabis" as an alternative and more neutral name for the drug. Importantly, however, no one has tested whether calling the drug "cannabis" as opposed to "marijuana" actually has any effect on public opinion. Using an original survey experiment, we examine whether framing the drug as "marijuana" as opposed to "cannabis" shapes public attitudes across a range of related topics: support for legalization of the drug, moral acceptance of its use, tolerance of activities involving the drug, perceptions of the drug's harms, and stereotypes of its users. Throughout each of our tests, we find no evidence to suggest that the public distinguishes between the terms "marijuana" and "cannabis." We conclude with implications of our findings for debates over marijuana/cannabis policy and for framing in policy discourse more generally.

Enhanced tolerance of industrial hemp (Cannabis sativa L.) plants on abandoned mine land soil leads to overexpression of cannabinoids.

Industrial activities have a detrimental impact on the environment and health when high concentrations of pollutants are released. Phytoremediation is a natural method of utilizing plants to remove contaminants from the soil. The goal of this study was to investigate the ability of Cannabis sativa L. to sustainably grow and remediate abandoned coal mine land soils in Pennsylvania. In this study, six different varieties of industrial hemp (Fedora 17, Felina 32, Ferimon, Futura 75, Santhica 27, and USO 31) were grown on two different contaminated soil types and two commercial soils (Miracle-Gro Potting Mix and PRO-MIX HP Mycorrhizae High Porosity Grower Mix). Plants growing in all soil types were exposed to two environmental conditions (outside and in the greenhouse). Seed germination response and plant height indicated no significant differences among all hemp varieties grown in different soils, however on an average, the height of the plants grown in the greenhouse exceeded that of the plants grown outdoors. In addition, heavy metal analysis of Arsenic, Lead, Nickel, Mercury, and Cadmium was performed. The concentration of Nickel was 2.54 times greater in the leaves of hemp grown in mine land soil outdoors when compared to greenhouse conditions. No differences were found between expression of heavy metal transporter genes. Secondary metabolite analysis of floral buds from hemp grown in mine land soil displayed a significant increase in the total Cannabidiol content (2.16%, 2.58%) when compared to Miracle-Gro control soil (1.08%, 1.6%) for outdoors and in the greenhouse, respectively. Molecular analysis using qRT-PCR indicated an 18-fold increase in the expression of the cannabidiolic acid synthase gene in plants grown on mine land soil. The data indicates a high tolerance to heavy metals as indicated from the physiological and metabolites analysis.

Phenotypic plasticity influences the success of clonal propagation in industrial pharmaceutical Cannabis sativa.

The burgeoning cannabis market requires evidence-based science such that farmers can quickly and efficiently generate new plants. In part, horticultural operations are limited by the success of cloning procedures. Here, we measured the role of environmental conditions and cultivar identity on the success of generating long branch material with many meristems in planting stock (mothers) and in rooting success of stem-derived clones. To evaluate the influence of lighting treatments on the optimal production of branching mothers, four lighting conditions (Fluorescent High Output T5s [T5], Metal halide lamps [MH], Plasma lamps [PL], or Metal halide lamps augmented with far red LED lights [MH+FR]) were applied to two cultivars of container grown plants (Cannabis sativa L. 'Bubba Kush', 'Ghost Train Haze') grown in peat-based organic substrates in mylar grow tents. To evaluate the influence of lighting, cutting tool (secateurs or scalpels), and stem wounding (present/absent) on optimal rooting of stems, three lighting conditions (Fluorescent T8s, T5, PL) were applied to three cultivars of peat pellet grown plants (C. sativa L. 'Bubba Kush', 'Ghost Train Haze', 'Headband'). Mothers grown under T5 and MH (vs MH+FR) produced ~30% more meristems. However, growing mothers under MH+FR were 19% taller than mothers under T5, with ~25% longer internodes on dominant stems than plants under any other lighting condition. Canopies were denser under T5 because petiole length was ~30% shorter under T5 and fan leaves were longer and narrower under MH+FR and MH+FR and PL, respectively, than under other lighting conditions. Cultivar Ghost Train Haze stems rooted most frequently and most quickly. Wounded stems were 162% more likely to root than unwounded stems and rooted 1.5 days earlier. Our results will guide producers attempting to maximize the rate of clone production in licensed facilities; although results may differ among cultivars, where cultivars differed in their average phenotype as mother plants, and their propensity to root from cuttings, and the speed with which they produced those roots.

Women's perspectives about cannabis use during pregnancy and the postpartum period: An integrative review.

Cannabis is the most commonly used illicit drug among general and pregnant populations. Despite recommendations to abstain from cannabis use, its use is increasing during the perinatal period. In this integrative review, we aim to understand women's perspectives about the health aspects of perinatal cannabis use. The following databases were searched: MEDLINE, PsycInfo, EMBASE, and CINAHL. We included quantitative and qualitative studies with a primary focus on perinatal cannabis use. The methodological quality of the included studies was appraised using appropriate checklists. Extracted data were integrated into a single data matrix and iteratively compared across studies to summarize and synthesize the data. Full text of 33 citations was retrieved and reviewed of which 6 studies met the inclusion criteria. Women who continued to use cannabis during pregnancy often perceived no general or pregnancy-specific risk compared to nonusers. The uncertainty regarding adverse perinatal consequences, its perceived therapeutic effects, and lower costs of cannabis compared to that of cigarettes contributed to cannabis use. A lack of communication with health care providers regarding the health aspects of cannabis was evident. Women perceived this lack of counseling as an indication that adverse outcomes associated with cannabis use are not significant. Women's perceptions of health risks associated with cannabis use are important factors in the decision-making process regarding use or cessation, particularly as legal concerns are fading away. A discussion about health concerns surrounding cannabis use may influence women's perceptions of risk and help them to make informed choices.

High times for cannabis: Epigenetic imprint and its legacy on brain and behavior.

Extensive debates continue regarding marijuana (Cannabis spp), the most commonly used illicit substance in many countries worldwide. There has been an exponential increase of cannabis studies over the past two decades but the drug's long-term effects still lack in-depth scientific data. The epigenome is a critical molecular machinery with the capacity to maintain persistent alterations of gene expression and behaviors induced by cannabinoids that have been observed across the individual's lifespan and even into the subsequent generation. Though mechanistic investigations regarding the consequences of developmental cannabis exposure remain sparse, human and animal studies have begun to reveal specific epigenetic disruptions in the brain and the periphery. In this article, we focus attention on long-term disturbances in epigenetic regulation in relation to prenatal, adolescent and parental germline cannabinoid exposure. Expanding knowledge about the protracted molecular memory could help to identify novel targets to develop preventive strategies and treatments for behaviors relevant to neuropsychiatric risks associated with developmental cannabis exposure.