ABSTRACT
An efficient system of genetic transformation and plant regeneration was established in Rehmannia glutinosa Libosch. f. hueichingensis (Chao et Schih) Hsiao by infecting the segments of leaves, stems and petioles of young regenerated plantlets with Agrobacterium rhizogenes strain 15834. Hairy roots were produced directly from the wounded surface of the explants on hormone-free Murashige and Skoog (MS) medium after infection by A. rhizogenes. Transformed roots grew rapidly either on solid or on liquid 1/2 MS medium, and exhibited typical hairy root phenotypes. The highest transformation frequency of 46.7% was achieved by pre-treating the A. rhizogenes with 100 micromol/L acetosyringone at logarithmic phase (OD600 = 1.8). The calluses with 100% induction frequency were induced from hairy roots on 1/2 MS medium containing 0.2 mg/L KT and 3.0 mg/L 6-BA, from which the shoots with 51.49% differentiation frequency was produced. These shoots could take root at a percentage of 100% and develope into four transformed plantlets when transferred on 1/2 MS medium, which had differences in morphological characters such as dwarfing, shortened nodals and abundant literal branching roots, and which survived vigorously after transplantation. The content of catalpol in an transformed hairy root clone was 0.557 mg/g. FW by means of HPLC, 48.5% and 18% of that in fresh and dried Rehmannia root, respectively. PCR and Southern blot analyses confirmed that rolB gene (564 bp) of TL-DNA was inserted in the genome of transformed hairy roots and their regenerated plantlets. RT-PCR analysis and opine paper electrophoresis detection revealed that TR-DNA containing opine synthetase gene was integrated and expressed in the genome of transformed hairy roots and their regenerated plantlets.
