PRODUCTION OF Brugmansia PLANTS FREE OF Colombian datura virus BY in vitro RIBAVIRIN CHEMOTHERAPY

Brugmansia x candida Pers ‘Creamsickle’ plants produced by in vitro treatment with ribavirin, and no thermal therapy, remained polymerase chain reaction (PCR-) negative for Columbian datura virus (CDV) after one year. The plants were produced by establishing B. x candida ‘Creamsickle’ shoot cultures on autoclaved MS basal medium (Murashige and Skoog, 1962), sucrose 30 g/L, myo-inositol 100 mg/L, thiamine HCl 1 mg/L, pyridoxine HCl 1 mg/L, nicotinic acid 1 mg/L, glycine 2 mg/L, BAP 1.1 µM, pH 5.7, and bacteriological agar (USB Corporation, Cleveland, Ohio, USA) 9.0 g/L with 15 ml of medium per 25×100 mm flat-bottomed glass culture tubes with polypropylene caps (Magenta Corporation, Chicago, Illinois, USA). The cultures were maintained in a growth room illuminated by cool-white fluorescent lamps (26 µmol m −2 s −1 ), constant 27°C, and a 16h photoperiod. Four weeks after initiation, the cultures were transferred to the same medium in polypropylene capped glass tubes except that the BAP concentration was reduced to 0.5 µM. In vitro-derived shoots were excised and further dissected to 3-6 mm in length before transferring onto the same medium containing ribavirin at 0, 50, 87.5, or 100 mg/L; these shoots were cultured for 30 days. The ribavirin treated shoots were then transferred onto the multiplication medium without ribavirin for one subculture before being rooted in vitro on the same MS basal medium except with one half strength MS nitrogen salts and 3 µM IAA for four weeks followed by greenhouse acclimatization. In vitro-derived plants that expressed no CDV symptoms and tested PCR-negative one year after transfer to the greenhouse were produced over the entire range of 50-100 mg/L ribavirin tested. A single line, CS2 2 B, from these PCR-negative plants was selected for long-term assessment - this line remains symptom-free and