Effects of Temperature and Centrifugation Conditions on DNA Isolation From Different Leaf Ages In Aconitum Heterophyllum Wall. Ex Royle

Abstract

Aconitum heterophyllum Wall. ex Royle, commonly known as Atees, is a critically endangered medicinal herb endemic to the Northwestern Himalayan region, valued for its therapeutic alkaloid atisine. The extraction of high-quality genomic DNA from this species presents significant challenges due to elevated concentrations of polysaccharides, polyphenolic compounds and secondary metabolites inherent to high-altitude medicinal plants. This study systematically evaluated the effects of leaf developmental stage (young, middle-aged and mature) and centrifugation conditions on DNA yield and purity using a modified CTAB protocol. Samples were collected from Ghesh village, Chamoli district, Uttarakhand and DNA was extracted under varying parameters: 10,000 rpm at 25°C, 12,000 rpm at 30°C and 15,000 rpm at 35°C. Spectrophotometric and electrophoretic analyses revealed that young leaves produced the highest DNA yield (847 µg/mL) and optimal purity ratios (A₂₆₀/A₂₈₀ = 1.82, A₂₆₀/A₂₃₀ = 2.15) under 10,000 rpm at 25°C, whereas higher centrifugation speeds and temperatures resulted in reduced quality. Optimal DNA extraction was achieved at 10,000 rpm and 25°C, demonstrating that elevated centrifugation speeds and temperatures inversely correlate with DNA quality. The inclusion of 1% PVP and 0.2% β-mercaptoethanol in the CTAB buffer effectively mitigated interference from secondary metabolites. RAPD-PCR amplification using primer OPA-1 (CAGGCCCTTC) generated clear and distinct banding patterns (7-9 bands) from optimally extracted DNA, confirming its suitability for molecular applications. These findings establish a reproducible, cost-effective protocol for high-quality genomic DNA extraction from A. heterophyllum, providing methodological guidance for molecular characterization, genetic diversity assessment and conservation strategies for endangered Himalayan medicinal flora.