An Optimized CTAB-Based Protocol for High-Quality Chloroplast DNA Isolation and PCR Validation in Dendrobium Hybrids

The study aimed to develop and standardize a reliable, cost-effective, and reproducible protocol for isolating chloroplast DNA (cpDNA) from two Dendrobium hybrids— Dendrobium ‘Sonia Earsakul’ and Dendrobium ‘Singapore White’ —and to assess its suitability for downstream molecular applications, such as PCR amplification using selected chloroplast markers. Leaf tissues were collected from healthy plants and subjected to three different DNA extraction methods: (i) a modified cetyltrimethylammonium bromide (CTAB) method, (ii) a modified sodium dodecyl sulfate (SDS) method, and (iii) the DNeasy Plant Mini Kit. The optimized CTAB protocol incorporated polyvinylpyrrolidone (PVP) and β-mercaptoethanol to counteract secondary metabolites present in orchid tissues. DNA yield and purity were measured using a Colibri + Microvolume UV-Vis spectrophotometer, while integrity was assessed by agarose gel electrophoresis. The extracted cpDNA was further validated through PCR amplification using three widely applied chloroplast markers: matK, trnL-F, and trnH-psbA. The standardized CTAB method produced the highest yield and purity, with DNA concentrations of 145–150 ng/µl, A260/A280 ratios of 1.88–1.92, and A260/A230 ratios of 2.03–2.11. In contrast, the SDS method and DNeasy kit produced lower yields and showed signs of contamination. Agarose gel electrophoresis confirmed intact, high-molecular-weight cpDNA in samples obtained by the CTAB protocol. Successful amplification of all three chloroplast markers confirmed the suitability of the isolated DNA for downstream molecular studies. The optimized CTAB-based protocol offers a robust, reproducible, and affordable method for cpDNA extraction from Dendrobium hybrids. Its superior performance over conventional and commercial methods demonstrates its potential utility in DNA barcoding, phylogenetics, breeding programs, and conservation of orchid genetic resources.