Transformation of ipecac (Cephaelis ipecacuanha) with Agrobacterium rhizogenes

Transformed root cultures of ipecac (Cephaelis ipecacuanha A. Richard), one of the recalcitrant woody plant species for Agrobacterium-mediated transformation, were established by co-culturing of in vitro petiole segments with Agrobacterium rhizogenes ATCC 15 834. Southern blot analysis of the established roots revealed that only the TL-DNA was integrated into the plant genome without incorporation of the TR-DNA. The transformed roots grew slowly on phytohormone-free solid medium and adventitious shoots were regenerated after over 6 months of culture on HF, half-strength Murashige and Skoog (1/2 MS) medium in the dark. The individually separated transformed shoots developed into plantlets on phytohormone-free solid medium at 25 degrees C under 16 h/day light, and the plants demonstrated wider leaves, shorter internodes and vigorous root growth compared to non-transformed plants. Effects of basal media and auxins on the growth and the ipecac alkaloid production of the transformed roots were investigated either under light or in the dark. The roots cultured in the dark grew well in Gamborg B5 (B5) liquid medium containing 0.5 mg/L IBA and yielded 112 mg/L of cephaeline and 14 mg/L emetine after 8 weeks of culture.