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.

Using a global diversity panel of Cannabis sativa L. to develop a near InfraRed-based chemometric application for cannabinoid quantification.

C. sativa has gained renewed interest as a cash crop for food, fibre and medicinal markets. Irrespective of the final product, rigorous quantitative testing for cannabinoids, the regulated biologically active constituents of C. sativa, is a legal prerequisite across the supply chains. Currently, the medicinal cannabis and industrial hemp industries depend on costly chromatographic analysis for cannabinoid quantification, limiting production, research and development. Combined with chemometrics, Near-InfraRed spectroscopy (NIRS) has potential as a rapid, accurate and economical alternative method for cannabinoid analysis. Using chromatographic data on 12 therapeutically relevant cannabinoids together with spectral output from a diffuse reflectance NIRS device, predictive chemometric models were built for major and minor cannabinoids using dried, homogenised C. sativa inflorescences from a diverse panel of 84 accessions. Coefficients of determination (r2) of the validation models for 10 of the 12 cannabinoids ranged from 0.8 to 0.95, with models for major cannabinoids showing best performance. NIRS was able to discriminate between neutral and acidic forms of cannabinoids as well as between C3-alkyl and C5-alkyl cannabinoids. The results show that NIRS, when used in conjunction with chemometrics, is a promising method to quantify cannabinoids in raw materials with good predictive results.

Semi-quantitative analysis of cannabinoids in hemp (Cannabis sativa L.) using gas chromatography coupled to mass spectrometry.

BACKGROUND: Hemp (Cannabis sativa L.) is a producer of cannabinoids. These organic compounds are of increasing interest due to their potential applications in the medicinal field. Advances in analytical methods of identifying and quantifying these molecules are needed. METHOD: This study describes a new method of cannabinoid separation from plant material using gas chromatography-mass spectrometry (GC-MS) as the analytical tool to detect low abundance cannabinoids that will likely have implications for future therapeutical treatments. A novel approach was adopted to separate trichomes from plant material to analyse cannabinoids of low abundance not observed in raw plant extract. Required plant sample used for analysis was greatly reduced compared to other methods. Derivatisation method was simplified and deconvolution software was utilised to recognise unknown cannabinoid compounds of low abundance. RESULTS: The method produces well-separated spectra and allows the detection of major and minor cannabinoids. Ten cannabinoids that had available standards could be identified and quantified and numerous unidentified cannabinoids or pathway intermediates based on GC-MS spectra similarities could be extracted and analysed simultaneously with this method. CONCLUSIONS: This is a rapid novel extraction and analytical method from plant material that can identify major and minor cannabinoids using a simple technique. The method will be of use to future researchers seeking to study the multitude of cannabinoids whose values are currently not understood.

An extreme-phenotype genome-wide association study identifies candidate cannabinoid pathway genes in Cannabis.

Cannabis produces a class of isoprenylated resorcinyl polyketides known as cannabinoids, a subset of which are medically important and exclusive to this plant. The cannabinoid alkyl group is a critical structural feature that governs therapeutic activity. Genetic enhancement of the alkyl side-chain could lead to the development of novel chemical phenotypes (chemotypes) for pharmaceutical end-use. However, the genetic determinants underlying in planta variation of cannabinoid alkyl side-chain length remain uncharacterised. Using a diversity panel derived from the Ecofibre Cannabis germplasm collection, an extreme-phenotype genome-wide association study (XP-GWAS) was used to enrich for alkyl cannabinoid polymorphic regions. Resequencing of chemotypically extreme pools revealed a known cannabinoid synthesis pathway locus as well as a series of chemotype-associated genomic regions. One of these regions contained a candidate gene encoding a ß-keto acyl carrier protein (ACP) reductase (BKR) putatively associated with polyketide fatty acid starter unit synthesis and alkyl side-chain length. Association analysis revealed twenty-two polymorphic variants spanning the length of this gene, including two nonsynonymous substitutions. The success of this first reported application of XP-GWAS for an obligate outcrossing and highly heterozygote plant genus suggests that this approach may have generic application for other plant species.

Complex Patterns of Cannabinoid Alkyl Side-Chain Inheritance in Cannabis.

The cannabinoid alkyl side-chain represents an important pharmacophore, where genetic targeting of alkyl homologs has the potential to provide enhanced forms of Cannabis for biopharmaceutical manufacture. Delta(9)-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) synthase genes govern dicyclic (CBDA) and tricyclic (THCA) cannabinoid composition. However, the inheritance of alkyl side-chain length has not been resolved, and few studies have investigated the contributions and interactions between cannabinoid synthesis pathway loci. To examine the inheritance of chemical phenotype (chemotype), THCAS and CBDAS genotypes were scored and alkyl cannabinoid segregation analysed in 210 F2 progeny derived from a cross between two Cannabis chemotypes divergent for alkyl and cyclic cannabinoids. Inheritance patterns of F2 progeny were non-Gaussian and deviated from Mendelian expectations. However, discrete alkyl cannabinoid segregation patterns consistent with digenic as well as epistatic modes of inheritance were observed among F2 THCAS and CBDAS genotypes. These results suggest linkage between cannabinoid pathway loci and highlight the need for further detailed characterisation of cannabinoid inheritance to facilitate metabolic engineering of chemically elite germplasm.

Multi-functionPlantDefensin,AntimicrobialandHeavyMetal Adsorbent Peptide.

BACKGROUND: Defensin peptide isolated from plants are often heterogeneous in length, sequence and structure, but they are mostly small, cationic and amphipathic. Plant defensins exhibit broad-spectrum antibacterial and antifungal activities against Gram-positive and Gram-negative bacteria, fungi and etc. Plant defensins also play an important role in innate immunity, such as heavy metal and some abiotic stresses tolerance. OBJECTIVES: In this paper, in vitro broad-spectrum activities, antimicrobial and heavy metal absorption, of a recombinant plant defensin were studied. MATERIAL AND METHODS: SDmod gene, a modified plant defensin gene, was cloned in pBISN1-IN (EU886197) plasmid, recombinant protein was produced by transient expression via Agroinfiltration method in common bean. The recombinant protein was tested for antibacterial activity against Gram-negative, Gram-positive bacteria and Fusarium sp. the effects of different treatments on heavy metal zinc absorption by this peptide were tested. RESULTS: We confirmed the antibacterial activities of this peptide against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Bacillus cereus) bacteria, and antifungal activities of this peptide against Fusarium spp. (Fusarium oxysporum and Fusarium solani). High metal absorption coefficient for this peptide was also observed. RESULTS: Out of six actinobacterial isolates, VITVAMB 1 possessed the most efficient RO-16 decolorization property. It decolorized 85.6% of RO-16 (250 mg L-1) within 24hrs. Isolate VITVAMB 1 was identified to be Nocardiopsis sp. Maximum dye decolorization occurred at pH 8, temperature 35oC, 3% salt concentration and a dye concentration of 50 mg L-1. CONCLUSIONS: Results suggesting that modified defensin peptide facilitates a broader range of defense activities. dedefensins are an important part of the innate immune system in eukaryotes. These molecules have multidimensional properties that making them promising agents for therapeutic drugs.

Developmental Plasticity of the Major Alkyl Cannabinoid Chemotypes in a Diverse Cannabis Genetic Resource Collection.

Cannabis is a chemically diverse domesticated plant genus which produces a unique class of biologically active secondary metabolites referred to as cannabinoids. The affinity and selectivity of cannabinoids to targets of the human endocannabinoid system depend on alkyl side chain length, and these structural-activity relationships can be utilized for the development of novel therapeutics. Accurate early screening of germplasm has the potential to accelerate selection of chemical phenotypes (chemotypes) for pharmacological exploitation. However, limited attempts have been made to characterize the plasticity of alkyl cannabinoid composition in different plant tissues and throughout development. A chemotypic diversity panel comprised of 99 individuals from 20 Cannabis populations sourced from the Ecofibre Global Germplasm Collection (ecofibre.com.au and anandahemp.com) was used to examine alkyl cannabinoid variation across vegetative, flowering and maturation stages. A wide range of di-/tri-cyclic as well as C3-/C5-alkyl cannabinoid composition was observed between plants. Chemotype at the vegetative and flowering stages was found to be predictive of chemotype at maturation, indicating a low level of plasticity in cannabinoid composition. Chemometric cluster analysis based on composition data from all three developmental stages categorized alkyl cannabinoid chemotypes into three classes. Our results suggest that more extensive chemical and genetic characterization of the Cannabis genepool could facilitate the metabolic engineering of alkyl cannabinoid chemotypes.

Association analysis of resistance to cereal cyst nematodes (Heterodera avenae) and root lesion nematodes (Pratylenchus neglectus and P. thornei) in CIMMYT advanced spring wheat lines for semi-arid conditions.

To identify loci linked to nematode resistance genes, a total of 126 of CIMMYT advanced spring wheat lines adapted to semi-arid conditions were screened for resistance to Heterodera avenae, Pratylenchus neglectus, and P. thornei, of which 107 lines were genotyped with 1,310 DArT. Association of DArT markers with nematode response was analyzed using the general linear model. Results showed that 11 markers were associated with resistance to H. avenae (pathotype Ha21), 25 markers with resistance to P. neglectus, and 9 significant markers were identified to be linked with resistance to P. thornei. In this work we confirmed that chromosome 4A (~90-105 cM) can be a source of resistance to P. thornei as has been recently reported. Other significant markers were also identified on chromosomal regions where no resistant genes have been reported for both nematodes species. These novel QTL were mapped to chromosomes 5A, 6A, and 7A for H. avenae; on chromosomes 1A, 1B, 3A, 3B, 6B, 7AS, and 7D for P. neglectus; and on chromosomes 1D, 2A, and 5B for P. thornei and represent potentially new loci linked to resistance that may be useful for selecting parents and deploying resistance into elite germplasm adapted to regions where nematodes are causing problem.

Mixed model association mapping for fusarium head blight resistance in tunisian-derived durum wheat populations.

Sources of resistance to Fusarium head blight (FHB) in wheat are mostly restricted to Chinese hexaploid genotypes. The effort to incorporate the resistance from hexaploid wheat or wild relatives to cultivated durum wheat (Triticum turgidum L. var. durum Desf.) have not been successful in providing resistance to the level of the donor parents. In this study, we used 171 BC(1)F(6) and 169 BC(1)F(7) lines derived from crossing of four Tunisian tetraploid sources of resistance (Tun7, Tun18, Tun34, Tun36) with durum cultivars 'Ben,' 'Maier,' 'Lebsock,' and 'Mountrail' for association studies. The Tun18 and Tun7 FHB resistances were found to be comparable to the best hexaploid wheat sources. A new significant QTL for FHB resistance was identified on the long arm of chromosome 5B (Qfhs.ndsu-5BL) with both association and classical QTL mapping analysis. Linkage disequilibrium (LD) blocks extending up to 40 cM were evident in these populations. The linear mixed model considering the structure (Q or P) and the kinship matrix (K(T)) estimated by restricted maximum likelihood (REML) was identified as the best for association studies in a mixture of wheat populations from a breeding program. The results of association mapping analysis also demonstrated a region on the short arm of chromosome 3B as potentially linked to FHB resistance. This region is in proximity of major FHB resistance gene fhb1 reported in hexaploid wheat. A possibility of having susceptibility or suppressor of resistance gene(s) on durum wheat chromosome 2A was further confirmed in this material, explaining the problem in developing resistant genotypes without counter selection against this region.