Growth and proteolytic activity of hairy roots from Centaurea calcitrapa: effect of nitrogen and sucrose

Abstract

Centaurea calcitrapa hairy root cultures were established by infection with Agrobacterium rhizogenes strain LBA 9402. The liquid medium hairy root cultures exhibited a biomass doubling time of approximately 1.5 days in the 20 days exponential growth phase. The effect of the initial sucrose and nitrogen concentrations in biomass and proteinase production of the liquid medium cultures was studied. The highest values for both proteolytic activity and fresh weight were attained between 30 and 50 g/l of sucrose. A low ammonium:nitrate ratio favoured the development of hairy roots and proteolytic activity. The best results for both parameters in terms of nitrogen nutrition were obtained with nitrate (24.7 mM) as the only nitrogen source. The maximum proteolytic activity was found to be at pH 4.0, within the pH range for aspartic proteinases (APs), and the inhibition studies showed that only pepstatin A, specific for that class of enzymes, revealed a significant inhibitory effect. The C. calcitrapa aspartic proteinase (cenprosin) gene was detected in hairy roots using specific polymerase chain reaction (PCR) primers. The specific proteolytic activity present in hairy roots seems to be lower than the reported for flowers but similar to the existent in the untransformed roots and cell suspension cultures.