PREPARATION OF HIGH-MOLECULAR-WEIGHT GENOMIC DNA FROM NUCLEI OF WOODY-PLANTS

We have developed a rapid and reliable method for preparation of high molecular weight genomic DNA from sweet orange (Citrus sinensis) suita ble for subsequent digestion by rarely cuffing restriction enzymes and then separation by pulsed-field gel electrophoresis (PFGE). Methods p reviously described for preparation of plant DNA prior to PFGE involve d protoplast isolation, a procedure that can be inefficient and time-c onsuming for several plant species. Nuclei isolated from plant tissues were embedded into agarose blocks and treated to release DNA, which w as cleaved by restriction enzymes and then submitted to PFGE. One gram of fresh leaves gave approximately 15 mu g of high molecular weight g enomic DNA (> 2000 kbp). Within-gel hybridizations were used; instead of classical Southern blotting, and the resulting signals were adequat e when they were compared with those obtained with DNA prepared from c rude ground leaf tissues.