Optimization of DNA extraction from fresh and dried Pinus brutia needles using different homogenization methods: An approach for forensic botanical applications

Obtaining sufficient DNA from plant tissues is essential for molecular taxonomy, population genetics, conservation biology, and forensic botany. However, DNA extraction from conifer species remains challenging because their tissues contain high concentrations of polysaccharides, polyphenols, terpenoids, and resin compounds that may interfere with DNA isolation and downstream molecular analyses. This study aimed to evaluate the effects of sample condition and homogenization method on DNA concentration obtained from fresh and dried needles of Pinus brutia, one of the most widespread conifer species in Türkiye. Needle samples were collected from 24 individual trees in Çanakkale, Türkiye. Samples from 12 trees were processed fresh, whereas samples from the remaining 12 trees were air-dried for 20 days. Within each sample condition, samples were processed using two homogenization methods: mortar grinding followed by TissueLyser homogenization and blender homogenization. Genomic DNA was extracted using the i-genomic Plant DNA Extraction Mini Kit and quantified using a Qubit™ 4 Fluorometer with the dsDNA High Sensitivity Assay. Data were analyzed using two-way analysis of variance (ANOVA). Fresh samples showed higher DNA concentrations than dried samples, and mortar grinding followed by TissueLyser homogenization resulted in higher DNA concentrations than blender homogenization. Two-way ANOVA demonstrated significant main effects of sample condition and homogenization method on DNA concentration, as well as a significant interaction between the two factors. These findings indicate that both sample condition and homogenization method influence DNA concentration obtained from P. brutia needles and highlight the importance of sample-specific optimization of DNA extraction procedures for molecular plant research and forensic botanical applications.