Fifteen hairy root lines of Catharanthus roseus were compared for growth, alkaloid production and shoot bud regeneration potential in relation to their morphology and pattern of rol gene(s) integration. These lines could be classified into 10 distinct morphotypes on the basis of their thickness and branching behaviour. Twelve root lines showed the presence of both rolB and rolC and two indicated the integration of only rolC. Genes rolA, B and C were only detected in N5 line. The root lines did not differ significantly for their total alkaloids content (1.10-1.30% dry wt.) but showed variations in relative concentration of ajmalicine (0.007-0.08% dry wt.), serpentine (0.01-0.08% dry wt.) and catharanthine (0.01-0.04% dry wt.). Line PG2 with highest biomass accumulation (GI = 326.73) and line PI3 with highest catharanthine content were up-scaled in 5-l bioreactor wherein a 9-11 folds increment in biomass accumulation was obtained with two folds enhancement in ajmalicine (0.029 % dry wt.) in PI3 line and appearance of a major unknown alkaloid in PG2 line. Shoot buds were induced only in line N5 on a MS + 3.0 mg/l BAP and 2.0 mg/l NAA which in turn showed excessive rooting and frequent in vitro flowering. © 2012 Copyright Taylor and Francis Group, LLC.