PURIFICATION OF TOMATO YELLOW LEAF CURL GEMINIVIRUS

Attempts were made to find a good purification procedure for tomato ye llow leaf curl virus (TYLCV), a dangerous and continuously spreading w hitefly-transmitted geminivirus, up to now only partially purified. El ectron microscopy, serology and spectrophotometry were used to evaluat e different procedures. The scheme finally adopted was the following: collect leaves and stems from Nicotiana benthamiana graft-infected 45- 60 days previously (5-10 g/plant); homogenize with 0.5 M phosphate buf fer pH 6 containing 2.5 mM NaEDTA, 10 mM Na2SO3, 0.1% 2- mercaptoethan ol, 1% Triton X-100 and 0.1% Driselase (3-4 ml of buffer for each g of material); incubate overnight on ice with gentle agitation; filter; e mulsify with 15% cold chloroform; centrifuge at low speed; ultracentri fuge supernatant: resuspend pellets in 0.5 M phosphate buffer pH 7 con taining 2.5 mM NaEDTA; centrifuge at low speed; repeat resuspension of the pellets and low-speed centrifugation; ultracentrifuge the pooled supernatant on a Cs2SO4 gradient (e.g. for 5 h at 41,000 rpm); collect the virus band and dialyse or ultracentrifuge the virus. The virus yi eld was 5-10 mg per kg of tissue.