Long-term hairy root culture of Centella asiatica (Apiaceae) for enhanced biomass accumulation and centellosides production

Centella asiatica is a well-known natural rich source of centellosides that possess various medicinal characteristics such as memory enhancement, anti-inflammatory, antioxidant, and wound healing activities. Nowadays, bioprocessing through hairy roots culture (HRC) is considered a proper approach for obtaining valuable specialized compounds. In this study, two hairy root types of C. asiatica, including a wild type (A4), inoculated with Rhizobium (Syn. Agrobacterium) rhizogenes C58C1 (pRiA4) and a transgenic type by R. tumefaciens C58C1 (pRiA4) (pBIs SQS1) carrying the Arabidopsis thaliana squalene synthase (At-SQS) gene were developed in long-term and batch culture systems for biomass accumulation and centellosides production. The highest fresh weight (623.17 ± 11.57 g/L) and dry weight (42.73 ± 0.60 g/L) were obtained for the SQS hairy root line (HRL3) in the long-term period at the eighth week, while in the batch culture system (also maintained for 8 weeks), the peak biomass was observed at the fourth week with maximum FW (243.25 ± 0.22 g/L) and DW (15.60 ± 0.001 g/L). Similar results for the A4 root line were also obtained in the long-term culture at the eighth week. Madecassoside showed the highest accumulation in both culture systems. The highest accumulation (mg/g DW) of madecassoside (4.09 ± 0.04) and total centellosides (5.11 ± 0.134) was observed at the fourth week, asiaticoside (0.57 ± 0.049) at the fifth week, and madecassic acid (0.67 ± 0.024) and asiatic acid (0.65 ± 0.033) at the first week was observed in the long-term system of the HRL3. The production of madecassoside in the SQS hairy root line was 2.67-fold higher than that of the wild type (A4) hairy root line (HRL9). The present results are noteworthy and may be considered for future commercial centellosides production in large-scale bioreactor cultures.