In vitro shoot regeneration of Centranthus ruber DC from hairy roots induced by Agrobacterium rhizogenes, and determination of valepotriate content

ABSTRACT

Hairy roots were induced by infection with Agrobacterium rhizogenes strain LBA 9402 containing the plasmid 1855 from the valepotriate-producing medicinal plant Centranthus ruber. Plants were regenerated from callus derived from the hairy roots. The induction of shoot domes was obtained when hairy root calli, after a period of 3 months of dark incubation in MS (Murashige & Skoog, 1962) medium without growth regulators, were transferred for one week on the same medium under a 16-h light / 8-h dark photoperiod. Shoot regeneration increased with benzyladenine alone applied monthly at 2.5 µM concentration after the 3 months period of dark incubation. No bud formation was observed in untransformed tissues grown under the same light and hormonal conditions. Callus cultures of non-transformed plants was obtained by using leaves and roots as source material in the presence of 10.7 µM NAA and 1 µM kinetin as growth regulators, and 9 µM BA and 2.6 µM NAA for the next phase of shoot regeneration. BA and NAA together were not used for plant regeneration from transformed tissue. The morphological characteristics of the transgenic plants were analysed during two years of ex vitro growth. In the greenhouse, the transgenic plants showed pale pink flowers, heterostyly, leaves smaller than those of wild type plants, and a larger amount of roots. Roots of transgenic plants continued to produce valepotriates.

KEY WORDS:

• Agrobacterium rhizogenes
• Centranthus ruber DC
• transgenic plants
• valepotriates