The isolation and purification of DNA from Vitis vinifera L. plants and in vitro cultures

Grapevine is a woody perennial , yet despite its economic importance, the mo1ecttl ar bio1ogy of Vitis is not well developed for its biotechno1ogical improvement (MULLINS 1990) . Although molecttlar studies have been reported (THOMAS et al. 1993), this study compares several procedures for the isolation ofDNA from glasshouse grown p1ants, and in vitro p1antlets and callus cultures. DNA was isolated from V vinifera L. cv. Sultanina (Thompson Seedless). The in vitro plantlets were estabIished (ROUBELAKJS-ANGELAKJS and ZIVANOVITIC 1991) and callus cultures were induced as described (KATSLRDAKIS and RouBELAKIS-ANGELAKJS 1991 ). The isolation of DNA from glasshouse grown grapevine p1ants was found to be problematic, and initial attempts at extracting total DNA using conventional procedures were unsuccessful. DNA preparations often became dark brown, presumably due to polyphenol oxidase activity from disrupted cell walls , vacuoles and organelles. Unknown compounds co-precipitated with the DNA to produce a viscous partially soluble product which reduced the yield ofDNA; thi s complex strongly absorbed UV light in the 230-280 nm region resulting in misleading estimates of DNA. The isolated DNA complex could not be separated in a caesium chloride-ethidium bromide density gradient; the aggregated material inhibited restriction enzymes and prevented analysis by agarase gel electrophoresis. ln view of these problems, a systematic study of DNA extraction procedures was performed. The use of cetyltrimethyl ammonium bromide (CTAB , see method 1, Table) to extract total DNA from grapevine was performed according to the method described by RoGERS and BENDICH ( 1988). The initial yields of DNA from g lasshouse plants were low due to an inability to precipitate DNA. This effect was attributed to V vin!fera 1eaf material having a relatively high mineral content and other unknown compounds (unpublished results) , which prevented the precipitation of the CTAB-DNA complex. This problem was solved by increasing the volume of precipitation buffer 3-4 fold, however the isolated DNA invariably turned black and inhibited restriction enzyme digestion. The use of phenol (method 2) to extract DNA from homogenised ti ssues followed by ethanol precipitation as described by FLAVELL et a/. ( 1988) produced a dark brown