Optimization of the DNA Extraction Protocol from the Leaves of Fritillaria eduardii Regel

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Abstract
Fritillaria eduardii Regel is a rare endemic species of high conservation and scientific value, yet molecular studies on this plant are often hampered by difficulties in DNA extraction due to the high content of polysaccharides, phenolic compounds, and secondary metabolites in its tissues. In this study, we tested and optimized a modified CTAB protocol for obtaining high-quality DNA from leaf tissues of F. eduardii. An initial protocol including polyvinylpyrrolidone (PVP) was evaluated, but the DNA yields and purity were inconsistent. Several key modifications were introduced, including exclusion of PVP from the composition, adjustment of buffer composition, replacement of chloroform–octanol with chloroform–isoamyl alcohol, the addition of a CTAB/NaCl purification step, and the use of a high-salt TE buffer for resuspension.Spectrophotometric analysis confirmed that the optimized protocol significantly improved DNA quality and stability. Among the initial nine test samples, only one fully met the optimal purity ratio (A260/A280 = 1.8–2.0), while others showed acceptable but suboptimal values, and three were unsuitable without additional purification. After protocol optimization, over 50 DNA samples were successfully obtained, with concentrations ranging from 548.7 to 4022.9 ng/µl and purity values between 1.80 and 2.09, confirming reproducibility and efficiency.The modified CTAB protocol provides reliable and reproducible DNA suitable for PCR amplification, sequencing, and phylogenetic studies. This approach can be recommended for F. eduardii and potentially adapted for other Fritillaria species rich in polysaccharides and phenolic compounds.

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