Transient studies of nutrient uptake, growth, and indole alkaloid accumulation in heterotrophic cultures of hairy roots ofCatharanthus roseus

Abstract

The kinetics of growth, the uptake of macronutrients, and the accumulation of indole alkaloids were investigated in long-term, heterotrophically cultured transgenic ("hairy") roots of Catharanthus roseus.Tabersonine, ajmalicine, and serpentine were monitored over a 70-day period. The doubling time [dry-weight (DW) basis] of C. roseus hairy roots in B5/2 nutrients supplemented with 3% sucrose was 3.6 days. NH(4)(+), NO(3),(-) and P(i) were depleted sequentially from culture medium by hairy roots, while sugars remained undepleted. The growth-limiting nutrient was inorganic nitrogen, NH(4)(+) and NO(3)(-), with exponential-phase overall biomass yields of 34.1 and 5.0 g DW/g nutrient, respectively. Extracellular pH decreased to 4.8 in early exponential phase of culture growth from the initially adjusted value of 5.7, increased subsequently to a maximum of 7.7 in late exponential phase of growth coincident with the maximum of fresh weight (FW)/DW ratio, before decreasing to 5.5-5.0. The organic acids, pyruvate, formate, lactate, and succinate were excreted by hairy roots starting in late phase of exponential growth, possibly resulting in the late-culture pH decrease. Tabersonine accumulation was distinctly growth associated with maximum specific and total yields of 1.15 mg/g DW and 5.6 mg/L, respectively, in late-exponential phase of growth. Serpentine accumulation was non growth associated with increasing specific and total levels in stationary growth phase: 1.3 mg/g DW and 10.5 mg/L, respectively. The accumulation of ajmalicine also appeared growth associated.