Abstract:A method of isolating megabase DNA from plant tissue was studied. Generally it includes isolation of purified nuclei by differential centrifugation, embedment of the nuclei in either low-melting-point agarose plugs or microbeads, and digestion with proteinase K in situ to release high molecular weight (HMW) DNA. The results indicated that the size of the prepared HMW nuclear DNA were influenced by both growth period of plant materials and the ways of embedment. The optimal condition for preparing HMW nuclear DNA was to isolate nuclei from the etiolated seedlings or young green leaves and to embedment the nuclei in agarose plugs. The DNA in one plug prepared in this best condition was between 200 kb and 5.7 Mb in size, mostly in the size of 2.2～5.7 Mb, and approximately 18～20 μg in amount. Since most of the organellar DNA was removed, the quality of HMW nuclear DNA prepared by this method was greatly improved comparing to those prepared by embedment of protoplast in agarose. The prepared DNA by this method was ready for partial and complete digestion with restriction enzymes and the digestion result was reproducible. The method is very simple and suitable for a wide range of plant taxa. The authors have used this method in isolating HMW nuclear DNA from rice ( Oryza sativa L. ), soybean ( Glycine max ( L. ) Merr. ), apple ( Malus pumila Mill. ), and com ( Zea mays L. ) successfully. The HMW nuclear DNA thus prepared facilitates plant genome analysis by pulsed field gel electrophoresis (PFGE) and construction of yeast and bacterial artificial chromosomal libraries.