Dataset Information

Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study.

ABSTRACT: Cannabis sativa is relatively recalcitrant to de novo regeneration, but several studies have reported shoot organogenesis or somatic embryogenesis from non-meristematic tissues. Most report infrequent regeneration rates from these tissues, but a landmark publication from 2010 achieved regeneration from leaf explants with a 96% response rate, producing an average of 12.3 shoots per explant in a single drug-type accession. Despite the importance regeneration plays in plant biotechnology and the renewed interest in this crop the aforementioned protocol has not been used in subsequent papers in the decade since it was published, raising concerns over its reproducibility. Here we attempted to replicate this important Cannabis regeneration study and expand the original scope of the study by testing it across 10 drug-type C. sativa genotypes to assess genotypic variation. In our study, callus was induced in all 10 genotypes but callus growth and appearance substantially differed among cultivars, with the most responsive genotype producing 6-fold more callus than the least responsive. The shoot induction medium failed to induce shoot organogenesis in any of the 10 cultivars tested, instead resulting in necrosis of the calli. The findings of this replication study raise concerns about the replicability of existing methods. However, some details of the protocol could not be replicated due to missing details in the original paper and regulatory issues, which could have impacted the outcome. These results highlight the importance of using multiple genotypes in such studies and providing detailed methods to facilitate replication.

SUBMITTER: Monthony AS

PROVIDER: S-EPMC8363012 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BACKGROUND: Biofuels extracted from the seeds of Camelina sativa have recently been used successfully as environmentally friendly jet-fuel to reduce greenhouse gas emissions. Camelina sativa is genetically very close to Arabidopsis thaliana, and both are members of the Brassicaceae. Although public databases are currently available for some members of the Brassicaceae, such as A. thaliana, A. lyrata, Brassica napus, B. juncea and B. rapa, there are no public Expressed Sequence Tags (EST) or genomic data for Camelina sativa. In this study, a high-throughput, large-scale RNA sequencing (RNA-seq) of the Camelina sativa transcriptome was carried out to generate a database that will be useful for further functional analyses. RESULTS: Approximately 27 million clean "reads" filtered from raw reads by removal of adaptors, ambiguous reads and low-quality reads (2.42 gigabase pairs) were generated by Illumina paired-end RNA-seq technology. All of these clean reads were assembled de novo into 83,493 unigenes and 103,196 transcripts using SOAPdenovo and Trinity, respectively. The average length of the transcripts generated by Trinity was 697 bp (N50 = 976), which was longer than the average length of unigenes (319 bp, N50 = 346 bp). Nonetheless, the assembly generated by SOAPdenovo produced similar number of non-redundant hits (22,435) with that of Trinity (22,433) in BLASTN searches of the Arabidopsis thaliana CDS sequence database (TAIR). Four public databases, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-prot, NCBI non-redundant protein (NR), and the Cluster of Orthologous Groups (COG), were used for unigene annotation; 67,791 of 83,493 unigenes (81.2%) were finally annotated with gene descriptions or conserved protein domains that were mapped to 25,329 non-redundant protein sequences. We mapped 27,042 of 83,493 unigenes (32.4%) to 119 KEGG metabolic pathways. CONCLUSIONS: This is the first report of a transcriptome database for Camelina sativa, an environmentally important member of the Brassicaceae. We showed that C. savita is closely related to Arabidopsis spp. and more distantly related to Brassica spp. Although the majority of annotated genes had high sequence identity to those of A. thaliana, a substantial proportion of disease-resistance genes (NBS-encoding LRR genes) were instead more closely similar to the genes of other Brassicaceae; these genes included BrCN, BrCNL, BrNL, BrTN, BrTNL in B. rapa. As plant genomes are under long-term selection pressure from environmental stressors, conservation of these disease-resistance genes in C. sativa and B. rapa genomes implies that they are exposed to the threats from closely-related pathogens in their natural habitats.

Project description:BACKGROUND:Cultured epidermal stem cells (Epi-SCs) and skin-derived precursors (SKPs) were capable of reconstituting functional hair follicles after implantation, while the signaling pathways that regulate neogenic hair follicle formation are poorly investigated. In this study, we aimed to understand the interactions between Epi-SCs and SKPs during skin organoid formation and to uncover key signal pathways crucial for de novo hair follicle regeneration. METHODS:To track their fate after transplantation, Epi-SCs derived from neonatal C57BL/6 mice were labeled with tdTomato, and SKPs were isolated from neonatal C57BL/6/GFP mice. A mixture of Epi-SCs-tdTomato and SKPs-EGFP in Matrigel was observed under two-photon microscope in culture and after implantation into excisional wounds in nude mice, to observe dynamic migrations of the cells during hair follicle morphogenesis. Signaling communications between the two cell populations were examined by RNA-Seq analysis. Potential signaling pathways revealed by the analysis were validated by targeting the pathways using specific inhibitors to observe a functional loss in de novo hair follicle formation. RESULTS:Two-photon microscopy analysis indicated that when Epi-SCs and SKPs were mixed in Matrigel and cultured, they underwent dynamic migrations resulting in the formation of a bilayer skin-like structure (skin organoid), where Epi-SCs positioned themselves in the outer layer; when the mixture of Epi-SCs and SKPs was grafted into excisional wounds in nude mice, a bilayer structure resembling the epidermis and the dermis formed at the 5th day, and de novo hair follicles generated subsequently. RNA-Seq analysis of the two cell types after incubation in mixture revealed dramatic alterations in gene transcriptome, where PI3K-Akt signaling pathway in Epi-SCs was significantly upregulated; meanwhile, elevated expressions of several growth factors and cytokine potentially activating PI3K were found in SKPs, suggesting active reciprocal communications between them. In addition, inhibition of PI3K or Akt by specific inhibitors markedly suppressed the hair follicle regeneration mediated by Epi-SCs and SKPs. CONCLUSIONS:Our data indicate that the PI3K-Akt signaling pathway plays a crucial role in de novo hair follicle regeneration, and the finding may suggest potential therapeutic applications in enhancing hair regeneration.

Dataset Information

Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets