Growth and terpenoid indole alkaloid production in Catharanthus roseus hairy root clones in relation to left- and right-termini-linked Ri T-DNA gene integration

Abstract

Hairy root cultures of Catharanthus roseus var. Prabal were established by infecting the leaves with Agrobacterium rhizogenes agropine-type A4 strain. Two hundred and fifty independent root clones were evaluated for growth, morphology, number of integration of Ri T-DNA genes and alkaloid contents. On the basis of growth pattern, type of branching and number of lateral roots we were able to separate the hairy root clones into four categories. However based on the integration of the Ri T_L-DNA and T_R-DNA genes, there were only three different categories of independent hairy root clones-C1 (rolA&B ⁺/ags⁺), C2 (rolA&B ⁻/ags⁺) and C3 (rolA&B ⁺/ags⁻). Southern hybridization analysis revealed both single and multiple copies of T-DNA integration in the root clones. The accumulation of considerable amounts of the root-specific alkaloids ajmalicine and serpentine was observed in the presence of both the T_L-DNA and T_R-DNA genes (C1) and the T_L-DNA gene (C3) alone. Two rolA&B⁻ but ags⁺ clones (C2) accumulated much less or only very negligible amounts of ajmalicine. The possible role of the T_L-DNA and T_R-DNA genes on growth and alkaloid accumulation in these root clones is discussed.