A simple and efficient CTAB plate-based protocol for genomic DNA extraction from crop plants

Abstract

Modern plant breeding and molecular genetics rely on genotyping large populations for applications such as genome editing and genomic selection. However, current DNA extraction methods often require expensive equipment or signif-icant manual labor, which limits their scalability. The objective of this study was to develop a scalable, cost-effective DNA extraction method. A bead-beating protocol was used to homogenize small amounts of leaf tissue from Arabidopsis, camelina, maize, sorghum, soybean, tobacco, and wheat, as well as developing soybean seeds. Samples were processed in 96-well plates using a standard CTAB (cetyltrimethylammonium bromide)-based extraction solution. DNA concentration was measured, and DNA quality was estimated using the A260/A280 and A260/A230 ratios and gel electrophoresis. The extracted DNA was then used for PCR assays to amplify targeted endogenous sequences, detect Cas9-edited sequences, and estimate gene copy numbers. This workflow enabled a single worker to process up to 960 samples per day. The total DNA yield was 2-3 {micro}g for leaf and 0.5-1 {micro}g for soybean seeds with good quality, which was sufficient for multiple PCR reactions. The DNA was successfully used for the intended PCR assays. The extracted DNA was suitable for reliable, downstream PCR-based genotyping. This method supports diverse analyses, including routine amplification, identification of genome edits, and copy number analysis.