Agrobacterium-mediated transformation of American ginseng with a rice chitinase gene

Transformation of American ginseng (Panax quinquefolius L.) with Agrobacterium strain LBA4404 containing a rice chitinase gene under control of the maize ubiquitin1 promoter and the phosphinothricin acetyltransferase (bar) and hygromycin phosphotransferase (hpt) genes as selectable markers is described. Epicotyl explants from 2- to 3-week-old ginseng seedlings were pre-cultured for 5–7 days on MS medium supplemented with 10 µM α-naphthaleneacetic acid and 9 µM 2,4-dichlorophenoxyacetic acid (ND medium) prior to Agrobacterium infection. The explants were either immersed in a bacterial suspension for 20 min or received a 10-µl or 15-µl droplet of bacteria. A co-culture period of 3 or 4 days was provided on ND medium with or without acetosyringone and ascorbic acid. Selection for transformed calli was conducted on ND medium containing 20 mg/l phosphinothricin or 100 mg/l hygromycin over a 10-month period. A maximum callusing frequency of 27.7% was achieved on selection medium when explants were infected by the droplet method and co-cultured on ND medium without acetosyringone and ascorbic acid. Almost 90% of the 32 lines that survived selection were shown to be transformed. Immersion of explants reduced the callusing frequency to 9.3%. The presence of the transgenes was detected by Southern hybridization and polymerase chain reaction (PCR) analysis. The expression of the chitinase gene was demonstrated by reverse transcription PCR and Western analysis. One hundred and two ginseng plantlets were recovered from 11 confirmed transgenic lines. The transgene integration in plantlets of two lines was demonstrated by Southern analysis. This is the first report of Agrobacterium-mediated transformation of this important medicinal plant.