Herbarized plants are a very valuable source of DNA, allowing the development of paleogenomics, the determination of evolutionary and phylogenetic relationships, and the determination of current ecosystem and global environmental changes. Plant cells differ from other organisms’ cells in a much more complex cellular structure and molecular composition. The cell wall in plants, consisting of polysaccharides, is challenging to break down, and secondary metabolites, proteins, and RNA are difficult to remove. An even greater amount of these substances is found in herbarium plants. Therefore, DNA isolated from herbarium plants has a much lower yield and higher contamination. For this reason, it is important to properly select the methodology for isolating DNA from herbarium plants, modify existing protocols, and optimize the DNA isolation process. Many methods exist for isolating DNA from herbarium plants, but it is still impossible to single out one of the most suitable ones. This work evaluated the suitability of several selected DNA extraction methods for DNA isolation from different herbarium plant species.
DNA from 6 different herbarium aquatic plants was isolated by 3 different methods: a modified CTAB method, Qiagen “Dneasy Plant Mini Kit,” and Thermo Fisher “GeneJET Plant Genomic DNA Purification Mini Kit.” Further modifications of the CTAB buffer were also performed: in one case, the amount of β-mercaptoethanol was doubled; in the second – the amount of PVP-40 was doubled. A Nanodrop spectrophotometer assessed the quality and concentration of the isolated DNA. ISSR-PCR amplification was performed with 4 selected primers (I-32, I-18, I-28, and Arcade-2) and visualized by agarose gel electrophoresis.
When comparing the three different methods, the highest DNA concentration was obtained by the CTAB method (CTAB – 249.04 ng/µL, Qiagen - 40.00 ng/µL, Fisher - 46.66 ng/µL), the absorbance ratios did not show significant differences.
ISSR-PCR amplification also did not show significant differences. After performing CTAB buffer modifications, higher DNA concentrations were obtained in most samples (β-mercaptoethanol – 222.15 ng/µL, PVP-40 336.78 ng/µL) and better A260/280 absorbance ratios (β-mercaptoethanol 1.97 ± 0.11, PVP-40 2.00 ± 0.06), and after performing ISSR-PCR amplification, sharper and clearer amplicon profiles were obtained.
The modifications made to the CTAB buffer improved the quality of DNA, increased its concentration, and allowed for higher-quality ISSR-PCR amplification results.