RESTRICTION ENDONUCLEASE ANALYSIS

Summary A major obstacle facing many taxonomists interested in analyzing variation at the DNA level is the preservation of their plant material, since plants are often collected far from laboratory facilities. We have studied effect of several commonly used preservation techniques on the quality of DNA isolated from preserved tissue. Chemical treatments (FAA, ethanol, Carnoy's solution, chloroformethanol) all result in DNA degradation; drying tissue, on the other hand, preserves DNA integrity, at least over a several month period. High molecular weight DNA suitable for restriction endonuclease digestion and analysis by DNA hybridization was isolated from dried leaves up to two years old. This suggests that recently-prepared dried specimens may represent an alternative to fresh tissue in molecular plant systematic studies. Studies involving DNA restriction fragment polymorphism analysis are becoming increasingly popular in plant systematics and population biology. Although such studies may utilize different molecules, such as chloroplast DNA (cf. Palmer et al., 1983; Sytsma and Gottlieb, 1986; Banks and Birky, 1985) or nuclear ribosomal genes (cf. Doyle and Beachy, 1985; Sytsma and Schaal, 1985; Saghai-Maroof et al., 1984), they have in common the need for high molecular weight DNA of good purity. Additionally, both systematists and population biologists need to be able to analyze a relatively large number of samples, a requirement that was, in the past, difficult to meet due to the expensive and time-consuming nature of DNA isolation techniques. This latter impediment has largely been overcome by advances in DNA isolation technology, with several different inexpensive "mini-prep" techniques having been published in recent years. These procedures all give high yields of DNA suitable for use in restriction endonuclease studies and require only small amounts of material (Saghai-Maroof et al., 1984; Rogers and Bendich, 1985; Appels and Dvorak, 1982). Despite these advances, a major problem still exists for researchers doing field work far from laboratory facilities, since most of these procedures are described for fresh or lyophilized tissue. DNA is present in dried plant material, as has been shown by Rogers and Bendich (1985); however, all of the herbarium specimens they used contained only relatively low molecular weight DNA that would, for the most part, not be useful in the types of studies that depend on the analysis of large restriction fragments. The purpose of our study was to investigate methods for preserving plant material for DNA analysis, with an eye toward finding a simple and efficient method of preservation that could be utilized by researchers in the field. Our particular concern is to develop a method useful for systematists who spend months on collecting trips in the tropics, but who wish to analyze variation at the DNA level as part of their research. Because such researchers are already using a variety of preservation methods to collect specimens for herbarium, anatomical and cytological investigation, our studies focused on testing several standard procedures for their suitability in preserving samples for DNA work.