Proc. Internl. Symp. on Technologies to Improve Sugar Productivity in Developing Countries, Guilin, P.R. China, pp 330-332 (2008) HIGH QUALITY GENOMIC DNA IMPURITIES-FREE FROM SUGAR CROPS AND OTHER PLANT TISSUE K. A. KHALED 1 and A. M. H. ESH 2 1 Breeding & Genetics Department, Sugar Crops Research Institute (SCRI), Agriculture Research Center (ARC), Giza, Egypt. 2 Plant protection Department, Sugar Crops Research Institute (SCRI), Agriculture Research Center (ARC), Giza, Egypt. Summary Aims: Polyphenolic compounds and polysaccharides are very important in DNA extraction and causes serious problems during the isolation of plant DNA especially in sugar crops (sugarbeet, sugarcane and sweet sorghum). Aim of this study is to develop a protocol for DNA extraction from fresh tissue that is applicable on plants containing high levels of polyphenols and polysaccharides. Methods and Results: The protocol described in this paper is a modified CTAB (hexadecyltrimethylammonium bromide) method, which produce a high quality genomic DNA from sugarbeet, sweet sorghum, and sugarcane. The methods used to extract high quality genomic DNA from different plant tissues (sugarbeet, sugarcane, sweet sorghum, maize, soybeans and alfalfa.) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be perform in any laboratory. Significance of the study: The amount of tissue required by this method is about 100 mg. The quantity and the quality of the DNA extracted by this method were high enough to perform hundreds of PCR-based reactions and to be use in other uses in DNA research. Key words: Alfalfa, DNA isolation, maize, polyphenols-free DNA, sugar beet, sugarcane, soybeans, sweet sorghum INTRODUCTION Tissues of many plant species contain high levels of polysaccharides and polyphenolic compounds, which impose a major problem in the purification of plant DNA. During the cells disruption, these cytoplasmic compounds can contact with nuclei and other organelles (Loomis, 1974). In their oxidized forms, the polyphenols covalently bind to DNA that results in a brown color and making it useless for most research applications (Katterman and Shattuck, 1983; Guillemaut and Maréchal-Drouard, 1992). There is current need for a rapid and efficient procedure for plants having high polysaccharides and polyphenols such as sugarcane and sweet sorghum. It is necessary when hundreds of samples need to be analyzed such as in genome mapping and marker assisted selection (MAS) programs. The high purity of the DNA is essential for PCR and PCR-based techniques such as random amplified polymorphic DNA (RAPD), microsatellite, RFLP and amplified fragment length polymorphism (AFLP), for genome mapping and DNA fingerprinting. Various protocols have been described for DNA isolation from plants containing high amounts of polyphenols including extraction of DNA from isolated nuclei (Hamilton et al., 1972) and purification by cesium chloride following the classic plant DNA protocol (Murray and Thompson, 1980) using liquid nitrogen and/or freeze-drying (lypholization) of the tissue for the initial grinding. Method for DNA extraction from plants containing high concentration of polyphenols and polysaccharides (Doyle et al. 1987; Honeycutt et al. 1992) and many modifications of a CTAB DNA extraction protocols have been published (Porebski et al., 1997; Peterson et al., 1997). However, these methods give a low yield of DNA and the procedures are relatively long. In addition, expensive reagents and tedious processes used in these protocols, such as RNase, spermidine, proteinase K. Our studies are an attempt to develop a protocol for DNA extraction from fresh tissue that is applicable on plants containing high levels of polyphenols and polysaccharides. MATERIALS AND METHODS A fresh tissue from sugarcane meristem cylinder (after removing outer leaf sheaths) or leaf tissue from any other plant (100 mg) was homogenized rapidly with 400 µl of homogenization buffer (25 mM Tris, pH 8.0; 10 mM EDTA; 0.8 M NaCl and 2% CTAB) followed by adding 100 µl of 5% N-Lauroyl-Sarcosine, 100 µl of 10% PVP, 100 µl of 10 % CTAB and 5 µl Mercapto-Ethanol. The samples then mixed well by inversion and incubated