Most cited article - PubMed ID 23505619
Changes of DNA methylation and hydroxymethylation in plant protoplast cultures
Cannabis sativa L. is a plant of significant interest due to its high content of biologically active compounds, durable fibers, and bioeconomic potential. Despite recent progress in protoplast isolation, complete plant regeneration from cannabis protoplasts remains unachieved, highlighting gaps in protoplast-to-plant systems. This study reports the second successful establishment and partial regeneration of cannabis protoplast cultures, and investigates their molecular dynamics, marking a significant step forward. We demonstrated that the age of donor material is critical for the protoplast isolation, with the optimal source being 1-2-week-old leaves from in vitro-grown seedlings. Cultivation in a modified medium developed for Arabidopsis thaliana supported initial cell divisions and microcallus formation. Transcriptomic analyses of cell proliferation and stress response markers indicate that the cultured protoplasts were viable, re-entered the cell cycle, and exhibited oxidative and abiotic stress resilience. These findings enhance our understanding of cannabis cell biology and lay the groundwork for a protoplast-based regeneration system, paving the way for advanced applications in biotechnology.
- Keywords
- Cannabis culture, cell proliferation, gene expression, in vitro, microcallus formation, oxidative stress, protoplast isolation, stress response,
- Publication type
- Journal Article MeSH
Epigenetic modifications play a vital role in the preservation of genome integrity and in the regulation of gene expression. DNA methylation, one of the key mechanisms of epigenetic control, impacts growth, development, stress response and adaptability of all organisms, including plants. The detection of DNA methylation marks is crucial for understanding the mechanisms underlying these processes and for developing strategies to improve productivity and stress resistance of crop plants. There are different methods for detecting plant DNA methylation, such as bisulfite sequencing, methylation-sensitive amplified polymorphism, genome-wide DNA methylation analysis, methylated DNA immunoprecipitation sequencing, reduced representation bisulfite sequencing, MS and immuno-based techniques. These profiling approaches vary in many aspects, including DNA input, resolution, genomic region coverage, and bioinformatics analysis. Selecting an appropriate methylation screening approach requires an understanding of all these techniques. This review provides an overview of DNA methylation profiling methods in crop plants, along with comparisons of the efficacy of these techniques between model and crop plants. The strengths and limitations of each methodological approach are outlined, and the importance of considering both technical and biological factors are highlighted. Additionally, methods for modulating DNA methylation in model and crop species are presented. Overall, this review will assist scientists in making informed decisions when selecting an appropriate DNA methylation profiling method.
