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Background Future breeding and selection of Cannabis sativa L. for both drug production and industrial purposes require a source of germplasm with wide genetic variation, such as that found in wild relatives and progenitors of highly cultivated plants. Limited directional selection and breeding have occurred in this crop, especially informed by molecular markers. Results This study investigated the population genomics of a natural cannabis collection comprising male and female individuals from various climatic zones in Iran. Using Genotyping-By-Sequencing (GBS), we sequenced 228 individuals from 35 populations. The data obtained enabled an association analysis, linking genotypes with key phenotypes such as inflorescence characteristics, flowering time, plant morphology, tetrahydrocannabinol (THC) and cannabidiol (CBD) content, and sex. We detected approximately 23,266 significant high-quality Single Nucleotide Polymorphisms (SNPs), establishing associations between markers and traits. The population structure analysis revealed that Iranian cannabis plants fall into five distinct groups. Additionally, a comparison with global data suggested that the Iranian populations is distinctive and generally closer to marijuana than to hemp, with some populations showing a closer affinity to hemp. The GWAS identified novel genetic loci associated with sex, yield, and chemotype traits in cannabis, which had not been previously reported. Conclusion The study's findings highlight the distinct genetic structure of Iranian Cannabis populations. The identification of novel genetic loci associated with important traits suggests potential targets for future breeding programs. This research underscores the value of the Iranian cannabis germplasm as a resource for breeding and selection efforts aimed at improving Cannabis for various uses.

Colonization of plants by fungal endophytes can improve plant growth and can assist in adaptation to biotic and abiotic stresses. The fungal endophytes Metarhizium robertsii and Pochonia chlamydosporia were previously shown to improve hemp growth. Here, the impact of three fungal endophytes, M. robertsii , P. chlamydosporia as well as Trichoderma harzianum on hemp was investigated under treatment with 300 mM NaCl as a salinity stress and reduced watering volume as a drought stress. Plant growth parameters, a lipid oxidation indicator, leaf porphyrins together with the abiotic stress responses genes were assessed in hemp with or without fungal colonization under normal and stressed conditions. Under salinity stress, the growth of hemp was ameliorated by the application of Metarhizium , Pochonia , or Trichoderma in the soil. The increased production of malondialdehyde (MDA) and the reduction in porphyrins in hemp under salinity stress were restored in the presence of fungal endophytes. Under drought stress, the aboveground growth of hemp was recovered by the application of Metarhizium together with the reduced production of porphyrins. The stress related gene CsNAC3 showed decreased expression during fungal application compared with uninoculated hemp under salinity or drought treatment. Colonization of Metarhizium , Pochonia or Trichoderma improved salt stress tolerance in hemp and this was accompanied by a reduction in oxidative stress.

Hemp ( Cannabis sativa L.) has a long cultivation history around the world. In northeast part of China, the alkaline soil geology severely reduces crop production. In this study, we tried to evaluate the impacts of alkali-induced stress on the photosynthetic status and physiological indices of hemp plants. The microscopic evaluation of endogenous ultrastructure clearly demonstrated significant oxidative damage to the structure of the photosynthetic tissues associated with the membrane, resulting from an increase in the levels of MGDG and DGDG. The deformed photosynthetic apparatus induced by alkali-stress significantly inhibited the biosynthesis process of photosynthetic pigments, causing 49.25%, 52.72%, 65.31%, and 28.13% loss in total Chl, Chl a, Chl b, and carotenoids, respectively. Meanwhile, the reduction in chlorophyll fluorescence parameters (Pn (74.62%), Gs (39.69%), and Tr (83.77%)) along with the obviously increased MDA (28.57%) and H 2 O 2 (35.18%) content exhibited that the inhibitory effect of alkali-stress not only decreased the photosynthetic efficiency by intercepting the nutrient supply but also generated excessive ROS, resulting in oxidative stress. Transcriptomic analysis (RNA-sequencing) revealed the considerably enriched GO terms as well as KEGG pathways that exposed the significant DEGs. The qPCR expression evaluation of down-regulated chlorophyll biosynthesis-related major genes ( GOGAT ( LOC115699366 ) and HEMA ( LOC133032634 )) and photosystem-related major genes ( PSB ( LOC115701338 ) and HCF ( LOC115707994 )) exhibited important molecular evidence for modulating the photosynthesis activity of hemp plant under devastating mechanism of alkali-stress. However, the transcript patterns of photorespiration-related genes ( GOX ( LOC115697365 ) and GDC ( LOC115707082 )) showed a slower decreasing trend at late stress stage (at 24 ~ 48 h), and the transcription of SGAT gene ( LOC115699360 ) was even enhanced by stress treatment at 48 h, probably in an attempt to adjust cellular carbon balance and elevate the antioxidant properties induced by alkali-stress.

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.

Background Protoplasts are a valuable tool for studying gene expression and applying genome editing techniques. Given the high medicinal and industrial potential of Cannabis sativa L., developing an efficient protoplast-to-plant regeneration protocol is highly desirable. Due to its recalcitrant nature, a complete plant regeneration from cannabis protoplasts has not yet been achieved. Results This study details a robust protocol for cannabis protoplast isolation, purification, transient transfection, and culture, additionally reporting somatic embryo-like structures derived from protoplast-derived callus. We demonstrated that the age of donor material, the composition of the enzyme solution, and the duration of enzymolysis are crucial for efficient protoplast isolation. Protoplast embedding, coupled with a rich culture medium and plant growth regulators, proved critical for initiating cell wall re-synthesis, cell division, and microcallus formation. Protoplasts isolated using the reported protocol were abundant (2.2 × 10 6 protoplasts/1 g of fresh weight), viable (78.8% viability) and able to undergo cell wall re-synthesis (56.1% of viable cells), followed by cell divisions (15.8% plating efficiency). Polyethylene glycol-mediated transfection yielded a 28% transfection efficiency and 17% plating efficiency in 10-day cultures. Protoplast-derived microcalli successfully proliferated on six regeneration media containing various concentrations of 6-benzylaminopurine and thidiazuron, exhibiting further proliferation and greening within two months. Conclusions This system provides a reliable protocol for isolation, transfection and culture of cannabis protoplasts. It also offers a framework for investigating gene function, as well as advancing protoplast fusion and genome editing technologies for this species.

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.

Cannabis sativa L. is an annual flowering herb of Eurasian origin that has long been associated with humans. Domesticated independently at multiple locations at different times for different purposes (food, fiber, and medicine), these long-standing human associations have influenced its distribution. However, changing environmental conditions and climatic fluctuations have also contributed to the distribution of the species and define where it is optimally cultivated. Here we explore the shifts in distribution that C. sativa may have experienced in the past and explore the likely shifts in the future. Modeling under paleoclimatic scenarios shows niche expansion and contraction in Eurasia through the timepoints examined. Temperature and precipitation variables and soil variable data were combined for species distribution modeling in the present day and showed high and improved predictive ability together as opposed to when examined in isolation. The five most important variables explaining ~ 65% of the total variation were soil organic carbon content (ORCDRC), pH index measured in water solution (PHIHOX), annual mean temperature (BIO-1), mean temperature of the coldest quarter (BIO-11) and soil organic carbon density (OCDENS) (AUC =  0.934). Climate model projections where efforts are made to curb emissions (RCP45/SSP245) and the business as usual (RCP85/SSP585) models were evaluated. Under projected future climate scenarios, shifts worldwide are predicted with a loss of ~ 43% in suitability areas with scores above 0.4 observed by 2050 and continued but reduced rates of loss by 2070. Changes in habitat range have large implications for the conservation of wild relatives as well as for the cultivation of Cannabis as the industry moves toward outdoor cultivation practices.

Background The breeding process enables plants to inherit desirable traits, such as yield, flowering time, pest resistance, and cannabinoid and/or terpene content. As a result of these intensive genetic improvement practices, where genetically similar individuals or those from the same lineage are crossed, the expression of unfavorable recessive alleles may occur due to homozygosity. This can lead to less productive plants, increased susceptibility to diseases, and reduced quality. Despite the potential negative effects associated with inbreeding, self-pollination (a form of inbreeding) is a necessary cultivation technique used to obtain seeds that produce phenotypically female plants (feminized seeds) for commercialization and/or to fix desirable traits, albeit at the cost of reduced genetic variability. The Cannabis sativa L. seed market has grown significantly in recent decades, driven by the legalization and regulation of medicinal and recreational use. Self-pollinated feminized seeds are popular among growers and commercial seed banks because, in most cases, they guarantee that inflorescences will express the cannabinoid and terpene profile of the single parent plant. The objective of this work is to compare the morphological variation of seeds obtained from the reversal of female clones followed by self-pollination, and seeds obtained from crossing genetically distinct parental. To study seed shape and size, we employed 2D geometric morphometrics (GM) based on landmarks and semilandmarks, coupled with a supervised machine learning approach and multivariate statistical approach for analysis. Results No direct relationship was observed between size and seed type, although significant differences between varieties were detected. The shape of seeds from crosses between different parents (male and female) showed lower classification accuracy compared to feminized seeds. These results support the hypothesis that inbreeding reduces the variability, as feminized seeds from self-pollination were correctly identified at a high rate using a discriminant function. Conclusions Our research demonstrates that 2D geometric morphometrics can effectively distinguish and trace feminized and self-pollinated cannabis seeds. These seeds exhibit the least morphological variation, enabling accurate identification and providing a reliable foundation for practical applications. The Random Forest classifier's high performance confirms the effectiveness of using morphological traits for seed discrimination. These results open the door for advanced machine-learning techniques aimed to improve scalability and automation.

While discrete regulatory mechanisms have been identified, a unified model for the transcriptional activation of photoperiodic flowering in short-day (SD) plants has not been delineated. Although the GIGANTEA-CONSTANS-FLOWERING LOCUS T ( GI-CO-FT ) pathway appears to be highly conserved, each species may differ in its blueprint for activation of FT and floral meristem identity. In cannabis, an SD plant, PSEUDO-RESPONSE-REGULATOR 37 (PRR37 ) has a causal effect in determining photoperiod sensitivity. This study identifies a network of WITH NO LYSINE (K) kinases ( WNK ) which may be regulating the PRR proteins and downstream genes, including florigen ( FT ). CO-like genes have been identified in cannabis, but their function in regulating FT has not been determined. This study shows that in cannabis, CO is critically-day-length-gated (CDL) and down-regulated by short days, likely to act as a repressor of FT . We demonstrate that CDL-gated gene expression can be manipulated in cannabis horticulture through modifying the R:FR ratio of LED lighting, where increased expression of COL5 in response to light treatment is inversely related with expression of FT . Building on these insights, we propose a model for the CDL-gated regulation of FT expression in cannabis.

Background Bama Huoma is a high-value cash crop because its seeds are known as the “holy seeds of longevity”. However, its seed yield is restricted by dioecism, and information about flower sex differentiation in this plant is lacking. Results In this study, anatomical analysis and dynamic transcriptome profiling were performed to explore the mechanism of sex differentiation in Bama Huoma. The results revealed that female and male flowers undergo independent developmental processes and that sex differentiation occurs at an early stage before flower development. Illumina sequencing revealed that MADS-box genes play crucial roles in pistil and stamen development. qRT‒PCR and RNA in situ hybridization were subsequently performed to determine the spatiotemporal expression patterns of genes related to sex determination. The results revealed that eight MADS-box genes and two MYB genes were expressed in a sex-biased and tissue-specific manner in male or female flowers. Among these genes, MYB35, PMADS2, TM6 and AGL104 were specifically expressed in male flowers and found to regulate callose metabolism, pollen sac formation, tapetum development, and microspore maturation. In contrast, AGL11, FBP24, AGL16 and two CAL were restricted to female flowers, where they regulate pistil formation, ovary development, and stigma differentiation. These genes play distinct and essential roles in the sex differentiation of Bama Huoma, as well as in the development of floral organs. Conclusion Our findings provide a comprehensive gene expression profile of flower sex differentiation in Bama Huoma as well as new clues and information for further study in this field.