Improved genomic DNA extraction from citrus species using a modified CTAB method.

BACKGROUND

Citrus species are recalcitrant to DNA extraction due to high levels of polysaccharides, polyphenols, and secondary metabolites that interfere with DNA yield and purity. This study presents an optimized cetyltrimethylammonium bromide (CTAB)-based protocol for efficient genomic DNA isolation from citrus leaves.

METHODS

Leaf tissues were ground in liquid nitrogen and extracted using a CTAB buffer supplemented with 2% β-mercaptoethanol and 2% polyvinylpyrrolidone. Contaminants were removed by two rounds of phenol: chloroform: isoamyl alcohol (25:24:1) extraction. RNase A treatment was applied, followed by DNA precipitation using cold isopropanol. DNA quality was assessed by A260/280 absorbance ratios and agarose gel electrophoresis. Statistical comparisons were made using the Mann-Whitney U test, Cohen's d, and bootstrap resampling.

RESULTS

The modified protocol yielded high-molecular-weight DNA with A260/280 ratios ranging from 1.78 to 1.98, indicating high purity. Electrophoresis confirmed intact DNA with minimal RNA contamination. DNA yield and band intensity were significantly higher than with the conventional method (p < 0.001, Cohen's d = -2.10). A positive correlation (r = 0.23) was observed between yield and band intensity. Bootstrap confidence intervals for mean band intensity were 0.11-0.20 (conventional) and 2.91-4.84 (modified).

CONCLUSION

The optimized CTAB protocol provides a simple, cost-effective, and reproducible method for high-quality genomic DNA extraction from citrus, suitable for PCR, genotyping, and sequencing applications.