Abstract:Pterocarpus marsupium is a medicinally important woody tree whose conservation, authentication and future biotechnological utilization can benefit from reliable molecular resources.. Obtaining usable DNA from woody medicinal plants can be technically challenging because leaf tissues contain polysaccharides, polyphenols and other secondary metabolites that may interfere with DNA recovery, purity and downstream enzymatic reactions. In the present work, several published DNA isolation approaches, including protocols associated with Healey et al. (2014), Michiels et al. (2002), and Doyle and Doyle (1987), were evaluated. Minor modifications were introduced, and the Doyle and Doyle-based CTAB approach produced the most consistent DNA quality. The optimized procedure incorporated pre-chilling, liquid-nitrogen grinding, β-mercaptoethanol-containing extraction buffer, optional polyvinylpyrrolidone (PVP), controlled incubation at 65 °C, chloroform:isoamyl alcohol purification, RNase A treatment, salt-assisted ethanol precipitation, ethanol washing and resuspension in TE buffer. DNA quantity and quality were evaluated by NanoDrop UV/Vis spectrophotometry and 0.7% agarose gel electrophoresis. Twenty DNA samples were assessed using absorbance at 260 and 280 nm, A260/A280 ratio and calculated DNA quantity. The recorded table values showed quantities from 0.186 to 0.768 mg/ml and A260/A280 ratios from 1.545 to 1.886. The highest quantities occurred in samples T1, T2, T5, T10, T15, T16, T18 and T20, while lower quantities occurred in T3, T4, T6, T7, T8, T9, T12, T13, T14, T17 and T19. Clear, intact bands on agarose gel supported the integrity of the extracted DNA. The findings demonstrate that the modified CTAB procedure can provide DNA of useful quality from P. marsupium leaf tissue and establish a foundation for subsequent PCR-based marker analysis, genetic fidelity testing, genetic diversity studies and conservation-oriented molecular investigations.

Keywords:Pterocarpus marsupium; genomic DNA; CTAB; polyvinylpyrrolidone; β-mercaptoethanol; RNase A; A260/A280; agarose gel electrophoresis; molecular characterization; conservation genetics.