Comparing methods for controlled capture and quantification of pollen in Cannabis sativa
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ABSTRACT:
SUBMITTER: Wizenberg S
PROVIDER: S-EPMC7526430 | biostudies-literature | 2020 Sep
REPOSITORIES: biostudies-literature
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Project description:Pollen grains are male gametophytes, an ephemeral haploid generation of plants, that commonly engage in competition for a limited supply of ovules. Since variation in reproductive capabilities among male gametophytes may influence the direction and pace of evolution in populations, we must be able to quantify the relative fitness of gametophytes from different sires. To explore this, we estimated the relative fitness of groups of male gametophytes in a dioecious, wind-pollinated model system, Cannabis sativa, by characterizing the non-abortion rate (measured via chemical staining) and viability (measured via in vitro germination) of pollen from multiple sires. Pollen viability quickly declined within two weeks of anther dehiscence, and pollen stored under freezer conditions did not germinate regardless of storage time. In contrast, pollen non-abortion rates declined slowly and persisted longer than the lifetime of a sporophyte plant under both room temperature and freezer conditions. Pollen samples that underwent both viability and non-abortion rate analysis displayed no significant correlation, implying that researchers cannot predict pollen viability from non-abortion rates, nor infer male gametophytic fitness from a single measure. Our work demonstrates two independent, differential approaches to measure proxies of male fitness in C. sativa.
| S-EPMC9262209 | biostudies-literature
Project description:Cannabis sativa L. is cultivated globally for its cannabinoid-dense inflorescences. Commercial preference for sinsemilla has led to the development of methods for producing feminized seeds through cross-pollination of cosexual (masculinized) female plants. Although the induction of cosexuality in Cannabis plants is common, to date, no work has empirically tested how masculinization of female Cannabis plants impacts male flowering, pollen production, pollen fitness, and related life-history trade-offs. Here, we cultivated a population of Cannabis plants (CFX-2) and explored how the route to cosexuality (drought vs. chemical induction) impacted flowering phenology, pollen production, and pollen fitness, relative to unsexual male plants. Unisexual males flowered earlier and longer than cosexual plants and produced 223% more total pollen (F2,28 = 74.41, p < 0.001), but per-flower pollen production did not differ across reproductive phenotypes (F2,21 = 0.887, p = 0.427). Pollen viability was 200% higher in unisexual males and drought-induced cosexuals (F2,36 = 189.70, p < 0.001). Pollen non-abortion rates only differed in a marginally significant way across reproductive phenotypes (F2,36 = 3.00, p = 0.06). Here, we demonstrate that masculinization of female plants impacts whole-plant pollen production and pollen fitness in Cannabis sativa.
| S-EPMC10648298 | biostudies-literature
Project description:Pollen viability and storage is of great interest to cannabis breeders and researchers to maintain desirable germplasm for future use in breeding or for biotechnological and gene editing applications. Here, we report a simple and efficient cryopreservation method for long-term storage of Cannabis sativa pollen. Additionally, the bicellular nature of cannabis pollen was identified using DAPI (4',6-diamidino-2-phenylindole) staining. A pollen germination assay was developed to assess cannabis pollen viability and used to demonstrate that pollen collected from different principal growth stages exhibited differential longevity. Finally, a simple and efficient method that employs pollen combined with baked whole wheat flour and subsequent desiccation under vacuum was developed for the long-term cryopreservation of C. sativa pollen. Using this method, pollen viability was maintained in liquid nitrogen after four months, suggesting long-term preservation of cannabis pollen.
| S-EPMC7284845 | biostudies-literature
Project description:Even if a large amount of high-throughput functional genomic data exists, most researchers feature a strong background in molecular biology but lack advanced bioinformatics skills. In this work, publicly available gene expression datasets have been analyzed giving rise to a total of 40,224 gene expression profiles within different Cannabis tissues/developmental stages. The resource here proposed will provide researchers with a starting point for future investigations of Cannabis sativa.
2017-01-13 | GSE93201 | GEO
Project description:Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.
| S-EPMC5371325 | biostudies-literature
Project description:BackgroundAllergic sensitization to Cannabis sativa is rarely reported, but the increasing consumption of marijuana has resulted in an increase in the number of individuals who become sensitized. To date, little is known about the causal allergens associated with C sativa.ObjectiveTo characterize marijuana allergens in different components of the C sativa plant using serum IgE from marijuana sensitized patients.MethodsSerum samples from 23 patients with a positive skin prick test result to a crude C sativa extract were evaluated. IgE reactivity was variable between patients and C sativa extracts. IgE reactivity to C sativa proteins in Western blots was heterogeneous and ranged from 10 to 70 kDa. Putative allergens derived from 2-dimensional gels were identified.ResultsProminent IgE reactive bands included a 23-kDa oxygen-evolving enhancer protein 2 and a 50-kDa protein identified to be the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. Additional proteins were identified in the proteomic analysis, including those from adenosine triphosphate synthase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and luminal binding protein (heat shock protein 70), suggesting these proteins are potential allergens. Deglycosylation studies helped refine protein allergen identification and demonstrated significant IgE antibodies against plant oligosaccharides that could help explain cross-reactivity.ConclusionIdentification and characterization of allergens from C sativa may be helpful in further understanding allergic sensitization to this plant species.
| S-EPMC3726218 | biostudies-literature
Project description:Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The main phytochemicals that are found in this plant are represented by cannabinoids, flavones, and terpenes. Some biological activities of cannabinoids are known to be enhanced by the presence of terpenes and flavonoids in the extracts, due to a synergistic action. In the light of all the above, the present study was aimed at the multi-component analysis of the bioactive compounds present in fibre-type C. sativa (hemp) inflorescences of different varieties by means of innovative HPLC and GC methods. In particular, the profiling of non-psychoactive cannabinoids was carried out by means of HPLC-UV/DAD, ESI-MS, and MS². The content of prenylated flavones in hemp extracts, including cannflavins A and B, was also evaluated by HPLC. The study on Cannabis volatile compounds was performed by developing a new method based on headspace solid-phase microextraction (HS-SPME) coupled with GC-MS and GC-FID. Cannabidiolic acid (CBDA) and cannabidiol (CBD) were found to be the most abundant cannabinoids in the hemp samples analysed, while ?-myrcene and ?-caryophyllene were the major terpenes. As regards flavonoids, cannflavin A was observed to be the main compound in almost all the samples. The methods developed in this work are suitable for the comprehensive chemical analysis of both hemp plant material and related pharmaceutical or nutraceutical products in order to ensure their quality, efficacy, and safety.
| S-EPMC6222702 | biostudies-literature
Project description:Four crosses were made between inbred Cannabis sativa plants with pure cannabidiol (CBD) and pure Delta-9-tetrahydrocannabinol (THC) chemotypes. All the plants belonging to the F(1)'s were analyzed by gas chromatography for cannabinoid composition and constantly found to have a mixed CBD-THC chemotype. Ten individual F(1) plants were self-fertilized, and 10 inbred F(2) offspring were collected and analyzed. In all cases, a segregation of the three chemotypes (pure CBD, mixed CBD-THC, and pure THC) fitting a 1:2:1 proportion was observed. The CBD/THC ratio was found to be significantly progeny specific and transmitted from each F(1) to the F(2)'s derived from it. A model involving one locus, B, with two alleles, B(D) and B(T), is proposed, with the two alleles being codominant. The mixed chemotypes are interpreted as due to the genotype B(D)/B(T) at the B locus, while the pure-chemotype plants are due to homozygosity at the B locus (either B(D)/B(D) or B(T)/B(T)). It is suggested that such codominance is due to the codification by the two alleles for different isoforms of the same synthase, having different specificity for the conversion of the common precursor cannabigerol into CBD or THC, respectively. The F(2) segregating groups were used in a bulk segregant analysis of the pooled DNAs for screening RAPD primers; three chemotype-associated markers are described, one of which has been transformed in a sequence-characterized amplified region (SCAR) marker and shows tight linkage to the chemotype and codominance.
| S-EPMC1462421 | biostudies-other
Project description:Cannabis sativa is a global multi-billion-dollar cash crop with numerous industrial uses, including in medicine and recreation where its value is largely owed to the production of pharmacological and psychoactive metabolites known as cannabinoids. Often underappreciated in this role, the lipoxygenase (LOX)-derived green leaf volatiles (GLVs), also known as the scent of cut grass, are the hypothetical origin of hexanoic acid, the initial substrate for cannabinoid biosynthesis. The LOX pathway is best known as the primary source of plant oxylipins, molecules analogous to the eicosanoids from mammalian systems. These molecules are a group of chemically and functionally diverse fatty acid-derived signals that govern nearly all biological processes including plant defense and development. The interaction between oxylipin and cannabinoid biosynthetic pathways remains to be explored. Despite their unique importance in this crop, there has not been a comprehensive investigation focusing on the genes responsible for oxylipin biosynthesis in any Cannabis species. This study documents the first genome-wide catalogue of the Cannabis sativa oxylipin biosynthetic genes and identified 21 LOX, five allene oxide synthases (AOS), three allene oxide cyclases (AOC), one hydroperoxide lyase (HPL), and five 12-oxo-phytodienoic acid reductases (OPR). Gene collinearity analysis found chromosomal regions containing several isoforms maintained across Cannabis, Arabidopsis, and tomato. Promoter, expression, weighted co-expression genetic network, and functional enrichment analysis provide evidence of tissue- and cultivar-specific transcription and roles for distinct isoforms in oxylipin and cannabinoid biosynthesis. This knowledge facilitates future targeted approaches towards Cannabis crop improvement and for the manipulation of cannabinoid metabolism.
| S-EPMC10132601 | biostudies-literature
Project description:Molecular identification of mixed-species pollen samples has a range of applications in various fields of research. To date, such molecular identification has primarily been carried out via amplicon sequencing, but whole-genome shotgun (WGS) sequencing of pollen DNA has potential advantages, including (1) more genetic information per sample and (2) the potential for better quantitative matching. In this study, we tested the performance of WGS sequencing methodology and publicly available reference sequences in identifying species and quantifying their relative abundance in pollen mock communities. Using mock communities previously analyzed with DNA metabarcoding, we sequenced approximately 200Mbp for each sample using Illumina HiSeq and MiSeq. Taxonomic identifications were based on the Kraken k-mer identification method with reference libraries constructed from full-genome and short read archive data from the NCBI database. We found WGS to be a reliable method for taxonomic identification of pollen with near 100% identification of species in mixtures but generating higher rates of false positives (reads not identified to the correct taxon at the required taxonomic level) relative to rbcL and ITS2 amplicon sequencing. For quantification of relative species abundance, WGS data provided a stronger correlation between pollen grain proportion and sequence read proportion, but diverged more from a 1:1 relationship, likely due to the higher rate of false positives. Currently, a limitation of WGS-based pollen identification is the lack of representation of plant diversity in publicly available genome databases. As databases improve and costs drop, we expect that eventually genomics methods will become the methods of choice for species identification and quantification of mixed-species pollen samples.
| S-EPMC8601920 | biostudies-literature